Structured Review

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Steady-state kinetic parameters for mAOX1-4 with aromatic aldehydes as substrates containing a benzyl-group. Apparent steady-state kinetic parameters were recorded in 50 mM <t>Tris-HCl,</t> 200 mM NaCl, and 1 mM <t>EDTA</t> (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
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1) Product Images from "Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities"

Article Title: Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191819

Steady-state kinetic parameters for mAOX1-4 with aromatic aldehydes as substrates containing a benzyl-group. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Figure Legend Snippet: Steady-state kinetic parameters for mAOX1-4 with aromatic aldehydes as substrates containing a benzyl-group. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

Techniques Used: Variant Assay, Activity Assay

Steady-state kinetic parameters for mAOX1-4 with N-heterocyclic compounds as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Figure Legend Snippet: Steady-state kinetic parameters for mAOX1-4 with N-heterocyclic compounds as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

Techniques Used: Variant Assay, Activity Assay

Steady-state kinetic parameters for mAOX1-4 with aliphatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Figure Legend Snippet: Steady-state kinetic parameters for mAOX1-4 with aliphatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

Techniques Used: Variant Assay, Activity Assay

Steady-state kinetic parameters for mAOX1-4 with cinnamaldehyde-related compounds as substrates aromatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Figure Legend Snippet: Steady-state kinetic parameters for mAOX1-4 with cinnamaldehyde-related compounds as substrates aromatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

Techniques Used: Variant Assay, Activity Assay

2) Product Images from "Identification of Channel-lining Amino Acid Residues in the Hydrophobic Segment of Colicin Ia"

Article Title: Identification of Channel-lining Amino Acid Residues in the Hydrophobic Segment of Colicin Ia

Journal: The Journal of General Physiology

doi: 10.1085/jgp.200810042

Effect of trans MTSET on the macroscopic current through colicin Ia mutant N578C channels. The top trace shows the membrane current and the bottom trace shows the voltage, each as a function of time. Before the start of the record, DTT was added to the trans compartment to a final concentration of 5 μM and 1.0 μg N578C (along with 4.5 μg octylglucoside and DTT to 5 μM) was added to the cis compartment. We quickly opened on the order of 1,000 channels by stepping the membrane potential to +70 mV, and then switched it to +50 mV to establish a slower channel-opening rate. At the arrow, 200 μg MTSET was added to the trans compartment. This caused a decrease in current of ∼25%, demonstrating that residue N578C was accessible for reaction. Finally, we confirmed that the channels closed normally when the membrane potential was switched to −50 mV. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM HEPES, pH 7.2. (The membrane broke after colicin addition and was reformed before the start of the record; similar results were obtained with membranes that had not broken.)
Figure Legend Snippet: Effect of trans MTSET on the macroscopic current through colicin Ia mutant N578C channels. The top trace shows the membrane current and the bottom trace shows the voltage, each as a function of time. Before the start of the record, DTT was added to the trans compartment to a final concentration of 5 μM and 1.0 μg N578C (along with 4.5 μg octylglucoside and DTT to 5 μM) was added to the cis compartment. We quickly opened on the order of 1,000 channels by stepping the membrane potential to +70 mV, and then switched it to +50 mV to establish a slower channel-opening rate. At the arrow, 200 μg MTSET was added to the trans compartment. This caused a decrease in current of ∼25%, demonstrating that residue N578C was accessible for reaction. Finally, we confirmed that the channels closed normally when the membrane potential was switched to −50 mV. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM HEPES, pH 7.2. (The membrane broke after colicin addition and was reformed before the start of the record; similar results were obtained with membranes that had not broken.)

Techniques Used: IA, Mutagenesis, Concentration Assay

3) Product Images from "Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability"

Article Title: Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200316889

Antimicrobial activity of calcium-bound surfactant-associated proteins. Human surfactant was repeatedly washed by sedimentation in the presence of calcium, eluted with EDTA, purified by mannose-Sepharose affinity chromatography, and size-fractionated by fast protein liquid chromatography on a Superose 6 column. ( a ) Elution of proteins from the column was monitored by UV absorbance at a wavelength of 280 nm. ( b ) The SP-A and SP-D content of individual fractions was determined by Western analysis. ( c ) The antimicrobial activity of the original sample and selected fractions, including 8, 12, and the tenfold concentrate of fraction 21, was assessed by measurement of 3 H-uridine incorporation in E. coli K12 . Controls shown included protein-free ultrafiltrates (molecular weight cutoff of 10,000) of the most concentrated sample used in each set (designated F), and 2 mM EDTA, the highest possible concentration of EDTA in any sample. Data are mean ± SEM; n = 3; * P
Figure Legend Snippet: Antimicrobial activity of calcium-bound surfactant-associated proteins. Human surfactant was repeatedly washed by sedimentation in the presence of calcium, eluted with EDTA, purified by mannose-Sepharose affinity chromatography, and size-fractionated by fast protein liquid chromatography on a Superose 6 column. ( a ) Elution of proteins from the column was monitored by UV absorbance at a wavelength of 280 nm. ( b ) The SP-A and SP-D content of individual fractions was determined by Western analysis. ( c ) The antimicrobial activity of the original sample and selected fractions, including 8, 12, and the tenfold concentrate of fraction 21, was assessed by measurement of 3 H-uridine incorporation in E. coli K12 . Controls shown included protein-free ultrafiltrates (molecular weight cutoff of 10,000) of the most concentrated sample used in each set (designated F), and 2 mM EDTA, the highest possible concentration of EDTA in any sample. Data are mean ± SEM; n = 3; * P

Techniques Used: Activity Assay, Sedimentation, Purification, Affinity Chromatography, Fast Protein Liquid Chromatography, Western Blot, Molecular Weight, Concentration Assay

4) Product Images from "DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells"

Article Title: DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells

Journal: Protein & Cell

doi: 10.1007/s13238-013-0018-8

ATP promotes the formation of DSS1 adducts with cellular proteins . (A) After overnight incubation of DSS1-biotin (20 ng) with NEM-treated HeLa lysate (50 μg) at 4°C under the conditions indicated, the lysates were separated by SDS-PAGE and detected using streptavidin-HRP or WB with anti-ubiquitin or anti-actin antibody. The pentagram star represents that the HeLa lysate at L7 was denatured at 95°C for 10 min. ATP, 2 mmol/L; EDTA, 10 mmol/L; Bortezomib, 20 μmol/L; NEM, 25 mmol/L. (B) The manner of DSS1 adduct formation is ATP dose-dependent. The NEM-treated HeLa lysates were digested with the USP2 (1 μg) to remove the pre-existing ubiquitin from its substrates. Actin served as an equal loading control
Figure Legend Snippet: ATP promotes the formation of DSS1 adducts with cellular proteins . (A) After overnight incubation of DSS1-biotin (20 ng) with NEM-treated HeLa lysate (50 μg) at 4°C under the conditions indicated, the lysates were separated by SDS-PAGE and detected using streptavidin-HRP or WB with anti-ubiquitin or anti-actin antibody. The pentagram star represents that the HeLa lysate at L7 was denatured at 95°C for 10 min. ATP, 2 mmol/L; EDTA, 10 mmol/L; Bortezomib, 20 μmol/L; NEM, 25 mmol/L. (B) The manner of DSS1 adduct formation is ATP dose-dependent. The NEM-treated HeLa lysates were digested with the USP2 (1 μg) to remove the pre-existing ubiquitin from its substrates. Actin served as an equal loading control

Techniques Used: Incubation, SDS Page, Western Blot

5) Product Images from "Oxygen-Linked S-Nitrosation in Fish Myoglobins: A Cysteine-Specific Tertiary Allosteric Effect"

Article Title: Oxygen-Linked S-Nitrosation in Fish Myoglobins: A Cysteine-Specific Tertiary Allosteric Effect

Journal: PLoS ONE

doi: 10.1371/journal.pone.0097012

S-nitrosation increases O 2 affinity of salmon and trout Mbs but not of tuna Mb and is functionally equivalent to modification by N -ethylmaleimide. A) O 2 equilibrium curves for tuna and salmon Mb and Mb-SNO and B) O 2 equilibrium curves for trout Mb, Mb-NEM and Mb-SNO, as indicated, measured in 50 mM Tris, 0.5 mM EDTA, pH 8.3 at 20°C. Mb-SNO data are from [9] .
Figure Legend Snippet: S-nitrosation increases O 2 affinity of salmon and trout Mbs but not of tuna Mb and is functionally equivalent to modification by N -ethylmaleimide. A) O 2 equilibrium curves for tuna and salmon Mb and Mb-SNO and B) O 2 equilibrium curves for trout Mb, Mb-NEM and Mb-SNO, as indicated, measured in 50 mM Tris, 0.5 mM EDTA, pH 8.3 at 20°C. Mb-SNO data are from [9] .

Techniques Used: Modification

6) Product Images from "The Arabidopsis Chloroplastic NifU-Like Protein CnfU, Which Can Act as an Iron-Sulfur Cluster Scaffold Protein, Is Required for Biogenesis of Ferredoxin and Photosystem I W⃞"

Article Title: The Arabidopsis Chloroplastic NifU-Like Protein CnfU, Which Can Act as an Iron-Sulfur Cluster Scaffold Protein, Is Required for Biogenesis of Ferredoxin and Photosystem I W⃞

Journal: The Plant Cell

doi: 10.1105/tpc.020511

Gel Filtration Analysis Revealed a Dimeric Holo-State and a Predominantly Monomeric Apo-State of AtCnfU-V. (A) Gel filtration chromatograms of holo- and apo-AtCnfU-V. After incubation with (dotted lines) or without (solid lines) 10 mM EDTA or 1 mM dithionite (dashed lines) on ice for 1 h, purified AtCnfU-V (25 μg) was applied to a Superdex 75 column (Amersham Biosciences) and equilibrated with buffer containing 50 mM Hepes-KOH, pH 7.5, 150 mM KCl, and 5 mM DTT. Eluates were monitored simultaneously by absorbance at 280 nm (top), 330 nm (middle), and 420 nm (bottom) and divided into 12 fractions. Molecular mass marker proteins used were BSA (67 kD), ovalbumin (43 kD), myoglobin (18 kD), and aprotinin (6.5 kD). ABS, absorbance. (B) Fractions (3 to 12) obtained from each gel filtration chromatography analysis shown in (A) were analyzed by protein gel blotting using an anti-AtCnfU-V antibody.
Figure Legend Snippet: Gel Filtration Analysis Revealed a Dimeric Holo-State and a Predominantly Monomeric Apo-State of AtCnfU-V. (A) Gel filtration chromatograms of holo- and apo-AtCnfU-V. After incubation with (dotted lines) or without (solid lines) 10 mM EDTA or 1 mM dithionite (dashed lines) on ice for 1 h, purified AtCnfU-V (25 μg) was applied to a Superdex 75 column (Amersham Biosciences) and equilibrated with buffer containing 50 mM Hepes-KOH, pH 7.5, 150 mM KCl, and 5 mM DTT. Eluates were monitored simultaneously by absorbance at 280 nm (top), 330 nm (middle), and 420 nm (bottom) and divided into 12 fractions. Molecular mass marker proteins used were BSA (67 kD), ovalbumin (43 kD), myoglobin (18 kD), and aprotinin (6.5 kD). ABS, absorbance. (B) Fractions (3 to 12) obtained from each gel filtration chromatography analysis shown in (A) were analyzed by protein gel blotting using an anti-AtCnfU-V antibody.

Techniques Used: Filtration, Incubation, Purification, Marker, Chromatography

7) Product Images from "A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1"

Article Title: A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0172182

Probable interrelationship between differential electrophoretic mobility of Hpn and Ni 2+ binding. Hpn may not have a definite form in the absence of Ni 2+ (A). After denaturation (B), smaller amounts of SDS binding/stacking behavior/larger hydrodynamic radius as well as a combination of some or all of these conditions (C) might have resulted in slower migration on SDS-PAGE (scheme highlighted with yellow background). Ni 2+ -treated Hpn forms a more compact structure (D). Pictorial structure of metalated Hpn is drawn to explain the model. MALDI spectra showed a partial Ni 2+ bound form (E) in denatured SDS-PAGE. Altered binding of SDS (F) caused by replacement of protein-protein to protein-SDS contacts (inhibiting stacking behavior) and/or degree of compactness (or reduced hydrodynamic radius) may be key factors responsible for “metal gel-shift” (scheme highlighted with green background). β-ME, β-mercaptoethanol; EDTA, ethylene diaminetetraacetic acid.
Figure Legend Snippet: Probable interrelationship between differential electrophoretic mobility of Hpn and Ni 2+ binding. Hpn may not have a definite form in the absence of Ni 2+ (A). After denaturation (B), smaller amounts of SDS binding/stacking behavior/larger hydrodynamic radius as well as a combination of some or all of these conditions (C) might have resulted in slower migration on SDS-PAGE (scheme highlighted with yellow background). Ni 2+ -treated Hpn forms a more compact structure (D). Pictorial structure of metalated Hpn is drawn to explain the model. MALDI spectra showed a partial Ni 2+ bound form (E) in denatured SDS-PAGE. Altered binding of SDS (F) caused by replacement of protein-protein to protein-SDS contacts (inhibiting stacking behavior) and/or degree of compactness (or reduced hydrodynamic radius) may be key factors responsible for “metal gel-shift” (scheme highlighted with green background). β-ME, β-mercaptoethanol; EDTA, ethylene diaminetetraacetic acid.

Techniques Used: Binding Assay, Migration, SDS Page, Electrophoretic Mobility Shift Assay

Confirmation of “Metal gel-shift” mechanism. A. Effect of EDTA and Ni 2+ ion treatment on migration rate of recombinant Hpn in SDS-PAGE (polyacrylamide-gel 20%). Lane M, protein marker (GE Healthcare; MW from top to bottom: 97, 66, 45, 30, 20.1 and 14.4 kDa); lane 1 and 2, Hpn before and after boiling (3 min at 100°C), respectively; lanes 3 and 4, EDTA-treated Hpn without and with boiling, respectively; lanes 5 and 6, Ni 2+ -treated Hpn without and with boiling, respectively. B. The SDS-PAGE analysis of partially-metalated-Hpn (25 μM) treated with increasing concentration of Ni 2+ (1:0, 1:0.8, 1:1.2, 1:1.6, 1:2.0, 1:2.4, 1:2.8, 1:3.2, 1:3.6, 1:4.0, 1:6.0 and 1:8.0). Lane M is marker proteins standard from Nacalai Tesque (200, 116, 66, 45, 31, 21.5, 14.4, 6.5 kDa from top to bottom respectively). Equal volume of heat-denatured protein applied in each lane. C. Scheme used for MALDI-TOF-MS analysis of Hpn protein that was heat denatured in Laemmli buffer. MS data was measured for Hpn treated with or without Ni 2+ ion (1:6 mol equivalent ratios). Further, MS data for Hpn (with or without Ni 2+ ) treated in Laemmli buffer (before and after SDS-PAGE) was measured. Even though some interference due to adducts was observed in samples treated with Laemmli buffer or gel-eluted fractions, metalated peaks (showing Hpn-Ni 2+ complexes) were distinct. The occurrence of metalated peaks was observed only for Ni 2+ -treated Hpn in all the conditions.
Figure Legend Snippet: Confirmation of “Metal gel-shift” mechanism. A. Effect of EDTA and Ni 2+ ion treatment on migration rate of recombinant Hpn in SDS-PAGE (polyacrylamide-gel 20%). Lane M, protein marker (GE Healthcare; MW from top to bottom: 97, 66, 45, 30, 20.1 and 14.4 kDa); lane 1 and 2, Hpn before and after boiling (3 min at 100°C), respectively; lanes 3 and 4, EDTA-treated Hpn without and with boiling, respectively; lanes 5 and 6, Ni 2+ -treated Hpn without and with boiling, respectively. B. The SDS-PAGE analysis of partially-metalated-Hpn (25 μM) treated with increasing concentration of Ni 2+ (1:0, 1:0.8, 1:1.2, 1:1.6, 1:2.0, 1:2.4, 1:2.8, 1:3.2, 1:3.6, 1:4.0, 1:6.0 and 1:8.0). Lane M is marker proteins standard from Nacalai Tesque (200, 116, 66, 45, 31, 21.5, 14.4, 6.5 kDa from top to bottom respectively). Equal volume of heat-denatured protein applied in each lane. C. Scheme used for MALDI-TOF-MS analysis of Hpn protein that was heat denatured in Laemmli buffer. MS data was measured for Hpn treated with or without Ni 2+ ion (1:6 mol equivalent ratios). Further, MS data for Hpn (with or without Ni 2+ ) treated in Laemmli buffer (before and after SDS-PAGE) was measured. Even though some interference due to adducts was observed in samples treated with Laemmli buffer or gel-eluted fractions, metalated peaks (showing Hpn-Ni 2+ complexes) were distinct. The occurrence of metalated peaks was observed only for Ni 2+ -treated Hpn in all the conditions.

Techniques Used: Electrophoretic Mobility Shift Assay, Migration, Recombinant, SDS Page, Marker, Concentration Assay, Mass Spectrometry

Amino acid sequence, overexpression and purification of recombinant Hpn. Lane M, LMW protein marker standards (GE Healthcare; MW from top to bottom: 97, 66, 45, 30, 20.1 and 14.4 kDa); black arrows depicting apo-Hpn and white arrows showing probable Ni 2+ -bound Hpn protein in all panels. A. Amino acid sequence of Hpn. Histidine residues are highlighted in bold. Stretches of six and seven histidines are highlighted in green and pentapetide repeats (EEGCC) are underlined. B. SDS-PAGE of Hpn expression with or without Ni 2+ added in the culture (polyacrylamide-gel 20%). Pellets of 60 μl bacterial cultures were dissolved in 60 μl of 1X Laemmli buffer and boiled for 3 min at 100°C. Final volume of 15 μl loaded in each lane. C, D and E. Elution profile of purified Hpn checked by loading protein fractions on SDS-PAGE (polyacrylamide-gel 15%). Lanes 1 to 10, fractions of purified protein eluted with 400 mM imidazole (C). Elution profiles of desalted fractions of Hpn without EDTA treatment (D) and with EDTA-treatment (E) were analyzed. Equal volume of 2X Laemmli buffer was added to each eluted fraction and then boiled for 3 min at 100°C. Total 10 μl applied in each lane in C, D and E.
Figure Legend Snippet: Amino acid sequence, overexpression and purification of recombinant Hpn. Lane M, LMW protein marker standards (GE Healthcare; MW from top to bottom: 97, 66, 45, 30, 20.1 and 14.4 kDa); black arrows depicting apo-Hpn and white arrows showing probable Ni 2+ -bound Hpn protein in all panels. A. Amino acid sequence of Hpn. Histidine residues are highlighted in bold. Stretches of six and seven histidines are highlighted in green and pentapetide repeats (EEGCC) are underlined. B. SDS-PAGE of Hpn expression with or without Ni 2+ added in the culture (polyacrylamide-gel 20%). Pellets of 60 μl bacterial cultures were dissolved in 60 μl of 1X Laemmli buffer and boiled for 3 min at 100°C. Final volume of 15 μl loaded in each lane. C, D and E. Elution profile of purified Hpn checked by loading protein fractions on SDS-PAGE (polyacrylamide-gel 15%). Lanes 1 to 10, fractions of purified protein eluted with 400 mM imidazole (C). Elution profiles of desalted fractions of Hpn without EDTA treatment (D) and with EDTA-treatment (E) were analyzed. Equal volume of 2X Laemmli buffer was added to each eluted fraction and then boiled for 3 min at 100°C. Total 10 μl applied in each lane in C, D and E.

Techniques Used: Sequencing, Over Expression, Purification, Recombinant, Marker, SDS Page, Expressing

8) Product Images from "Stability and Function of the Sec61 Translocation Complex Depends on the Sss1p Tail-Anchor Sequence"

Article Title: Stability and Function of the Sec61 Translocation Complex Depends on the Sss1p Tail-Anchor Sequence

Journal: The Biochemical journal

doi: 10.1042/BJ20101865

Digitonin-soluble Sec complexes prepared from membranes from mutant Sss1p strains are unstable when assayed by binding to Concanavalin A Sepharose (A) Post-nuclear membranes derived from yeast expressing Sss1pWT, Sss1pYG, and Sss1pF were salt extracted, washed several times to remove EDTA, solubilized in either 2.5% digitonin or 1% Triton X-100 (wild type only), and pre-cleared by centrifugation to remove insoluble components. Soluble Sec complexes were bound to Concanavalin A Sepharose (Con A beads) which were separated from free Sec61 complexes by low speed centrifugation. Bound Sec61 complexes forming part of the Sec complex were then eluted from the Con A beads with 1% Triton X-100 and high salt. Samples were either acid precipitated or had loading buffer added (Con A beads) and were analyzed by SDS-PAGE and immunoblotting with antibodies to Sec63p, Sec61p, Sec72p, and Sss1p. Free , fraction of detergent-soluble proteins not bound to Concanavalin-A; Bound , proteins bound to Concanavalin-A and eluted with 1% Triton X-100 and 500mM NaCl; Beads , proteins still bound to Concanavalin-A after elution with 1% Triton and 500mM NaCl; * contaminant eluted from beads. (B) Experiments performed as described in (A) were quantified and expressed as percent bound to Con A for each protein indicated. WT T, Sss1pWT-derived post-nuclear membranes solubilized in 1% Triton X-100; WT D, YG D, F D, digitonin-solubilized post-nuclear membranes derived from strains expressing Sss1pWT, Sss1pYG, and Sss1pF, respectively.
Figure Legend Snippet: Digitonin-soluble Sec complexes prepared from membranes from mutant Sss1p strains are unstable when assayed by binding to Concanavalin A Sepharose (A) Post-nuclear membranes derived from yeast expressing Sss1pWT, Sss1pYG, and Sss1pF were salt extracted, washed several times to remove EDTA, solubilized in either 2.5% digitonin or 1% Triton X-100 (wild type only), and pre-cleared by centrifugation to remove insoluble components. Soluble Sec complexes were bound to Concanavalin A Sepharose (Con A beads) which were separated from free Sec61 complexes by low speed centrifugation. Bound Sec61 complexes forming part of the Sec complex were then eluted from the Con A beads with 1% Triton X-100 and high salt. Samples were either acid precipitated or had loading buffer added (Con A beads) and were analyzed by SDS-PAGE and immunoblotting with antibodies to Sec63p, Sec61p, Sec72p, and Sss1p. Free , fraction of detergent-soluble proteins not bound to Concanavalin-A; Bound , proteins bound to Concanavalin-A and eluted with 1% Triton X-100 and 500mM NaCl; Beads , proteins still bound to Concanavalin-A after elution with 1% Triton and 500mM NaCl; * contaminant eluted from beads. (B) Experiments performed as described in (A) were quantified and expressed as percent bound to Con A for each protein indicated. WT T, Sss1pWT-derived post-nuclear membranes solubilized in 1% Triton X-100; WT D, YG D, F D, digitonin-solubilized post-nuclear membranes derived from strains expressing Sss1pWT, Sss1pYG, and Sss1pF, respectively.

Techniques Used: Size-exclusion Chromatography, Mutagenesis, Binding Assay, Derivative Assay, Expressing, Centrifugation, SDS Page

9) Product Images from "Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading"

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033662

Tyrosine phosphorylation of cortactin terminates its interaction with focal adhesion kinase (FAK) during cell spreading. ( A ) Coomassie staining of purified GST and GST-cortactin SH3 domain was scanned in the Odyssey system. ( B ) HeLa cells were detached with trypsin-EDTA, washed with trypsin inhibitor and kept in suspension (susp.) or allowed to spread for 3 h on fibronectin (FN)-treated 100-mm plates. RIPA cell lysates were used for pull-down experiments with GST or GST-SH3, which were analyzed by SDS-PAGE and WB with focal adhesion kinase (FAK) Ab, followed by labeling with a 800CW-conjugated goat rabbit Ab. ( C ) HeLa cells were transfected with ZipB-MycCortactin and empty vector (TF2) or with ZipB-MycCortactin and ZipA-HAΔSrc (TF3). After 20 h cells were detached with trypsin-EDTA, washed with trypsin inhibitor and allowed to spread on FN-coated 100-mm plates for 3 h. Cell lysates were subjected to immunoprecipitation with FAK MoAb. The immunoprecipitates were subjected to WB and probed in three steps: (1) with myc Ab to detect transfected cortactin, followed by a 680CW-labeled goat mouseAb (red); (2) with FAK Ab, followed by a 800CW-labeled goat rabbit Ab (green); and (3) with pY466 cortactin Ab, followed by a 800CW-labeled goat rabbit Ab. Transfected cortactin was immunoprecipitated by FAK (asterisk) only when the protein was not tyrosine-phosphorylated.
Figure Legend Snippet: Tyrosine phosphorylation of cortactin terminates its interaction with focal adhesion kinase (FAK) during cell spreading. ( A ) Coomassie staining of purified GST and GST-cortactin SH3 domain was scanned in the Odyssey system. ( B ) HeLa cells were detached with trypsin-EDTA, washed with trypsin inhibitor and kept in suspension (susp.) or allowed to spread for 3 h on fibronectin (FN)-treated 100-mm plates. RIPA cell lysates were used for pull-down experiments with GST or GST-SH3, which were analyzed by SDS-PAGE and WB with focal adhesion kinase (FAK) Ab, followed by labeling with a 800CW-conjugated goat rabbit Ab. ( C ) HeLa cells were transfected with ZipB-MycCortactin and empty vector (TF2) or with ZipB-MycCortactin and ZipA-HAΔSrc (TF3). After 20 h cells were detached with trypsin-EDTA, washed with trypsin inhibitor and allowed to spread on FN-coated 100-mm plates for 3 h. Cell lysates were subjected to immunoprecipitation with FAK MoAb. The immunoprecipitates were subjected to WB and probed in three steps: (1) with myc Ab to detect transfected cortactin, followed by a 680CW-labeled goat mouseAb (red); (2) with FAK Ab, followed by a 800CW-labeled goat rabbit Ab (green); and (3) with pY466 cortactin Ab, followed by a 800CW-labeled goat rabbit Ab. Transfected cortactin was immunoprecipitated by FAK (asterisk) only when the protein was not tyrosine-phosphorylated.

Techniques Used: Staining, Purification, SDS Page, Western Blot, Labeling, Transfection, Plasmid Preparation, Immunoprecipitation

10) Product Images from "Tertiary and quaternary allostery in HbII from Scapharca inaequivalvis"

Article Title: Tertiary and quaternary allostery in HbII from Scapharca inaequivalvis

Journal: Biochemistry

doi: 10.1021/bi301620x

Oxygen binding curve to HbII in a solution containing 100 mM phosphate, 1 mM EDTA, pH 7.0, 15 °C (open circles). Data points were fitted to the Hill equation (dashed line) with a p50 of 7.73 ± 0.12 torr and a Hill coefficient of 1.90 ± 0.05, and to the Adair equation (solid line) with dissociation constants: K 1 = 0.0446 ± 0.0072 torr −1 , K 2 = 0.0502 ± 0.0268 torr −1 , K 3 = 0.1974 ± 0.1319 torr −1 , K 4 = 0.7524 ± 0.2348 torr −1 .
Figure Legend Snippet: Oxygen binding curve to HbII in a solution containing 100 mM phosphate, 1 mM EDTA, pH 7.0, 15 °C (open circles). Data points were fitted to the Hill equation (dashed line) with a p50 of 7.73 ± 0.12 torr and a Hill coefficient of 1.90 ± 0.05, and to the Adair equation (solid line) with dissociation constants: K 1 = 0.0446 ± 0.0072 torr −1 , K 2 = 0.0502 ± 0.0268 torr −1 , K 3 = 0.1974 ± 0.1319 torr −1 , K 4 = 0.7524 ± 0.2348 torr −1 .

Techniques Used: Binding Assay

Elution profiles of G-100 Sephadex size exclusion chromatography of solutions containing 100 μM (solid black line) and 2 μM HbA (dashed black line), and 107 μM (solid grey line) and 5.6 μM HbII (dashed grey line), carried out at 20 °C in 100 mM phosphate, 1 mM EDTA, pH 7.0.
Figure Legend Snippet: Elution profiles of G-100 Sephadex size exclusion chromatography of solutions containing 100 μM (solid black line) and 2 μM HbA (dashed black line), and 107 μM (solid grey line) and 5.6 μM HbII (dashed grey line), carried out at 20 °C in 100 mM phosphate, 1 mM EDTA, pH 7.0.

Techniques Used: Size-exclusion Chromatography

11) Product Images from "Structural Correlates of Rotavirus Cell Entry"

Article Title: Structural Correlates of Rotavirus Cell Entry

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004355

Internalization. A . Image of three particles that uncoat after addition of fluorescently labeled m159 antibody to the medium. (Virus pre-bound to cells, excess medium withdrawn, and antibody-containing medium then added.) Particles that uncoat do not bind antibody. White line represents edge of cell; particles on the coverslip all bind antibody. B . Time to initiation of uncoating (decrease in VP7 and VP4 label intensities) of particles that do not bind antibody m159. The antibody was added 5–7 minutes after addition of virus to cells, as indicated by the arrow; images were collected every 6 seconds. C . EDTA pulse. Top panel: doubly labeled virus bound to cell, before and after EDTA pulse. Middle panel: DIC images of the same cell; Bottom panel: particles bound to coverslip. D . Percent of virus resistant to EDTA uncoating, as a function of time between addition of virus and pulse of EDTA (black open circles) and cumulative representation of data in 2B (gray solid circles).
Figure Legend Snippet: Internalization. A . Image of three particles that uncoat after addition of fluorescently labeled m159 antibody to the medium. (Virus pre-bound to cells, excess medium withdrawn, and antibody-containing medium then added.) Particles that uncoat do not bind antibody. White line represents edge of cell; particles on the coverslip all bind antibody. B . Time to initiation of uncoating (decrease in VP7 and VP4 label intensities) of particles that do not bind antibody m159. The antibody was added 5–7 minutes after addition of virus to cells, as indicated by the arrow; images were collected every 6 seconds. C . EDTA pulse. Top panel: doubly labeled virus bound to cell, before and after EDTA pulse. Middle panel: DIC images of the same cell; Bottom panel: particles bound to coverslip. D . Percent of virus resistant to EDTA uncoating, as a function of time between addition of virus and pulse of EDTA (black open circles) and cumulative representation of data in 2B (gray solid circles).

Techniques Used: Labeling

VP4 fusion-loop mutant and VP7 disulfide-linked trimer. A . Effects of mutations on uncoating. Particles recoated with VP4 fusion-loop mutant (top panel) and VP7 disulfide-linker trimer (bottom panel) added to BSC-1 cells and imaged at 2 min and 45 min. All particles in the field at 45 sec retain VP7. B . Percent uncoating for particles recoated with wt proteins, with VP4 mutant and VP7 wt, and with VP4 wt and VP7 mutant. Average of three experiments; bars show standard error. C . Percent of particles protected from EDTA dissociation (for wt and VP4-391D) and from access by m159 (for VP7 C-C) as a function of time. Average of three experiments; bars show standard error.
Figure Legend Snippet: VP4 fusion-loop mutant and VP7 disulfide-linked trimer. A . Effects of mutations on uncoating. Particles recoated with VP4 fusion-loop mutant (top panel) and VP7 disulfide-linker trimer (bottom panel) added to BSC-1 cells and imaged at 2 min and 45 min. All particles in the field at 45 sec retain VP7. B . Percent uncoating for particles recoated with wt proteins, with VP4 mutant and VP7 wt, and with VP4 wt and VP7 mutant. Average of three experiments; bars show standard error. C . Percent of particles protected from EDTA dissociation (for wt and VP4-391D) and from access by m159 (for VP7 C-C) as a function of time. Average of three experiments; bars show standard error.

Techniques Used: Mutagenesis, Size-exclusion Chromatography

12) Product Images from "Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells *"

Article Title: Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.663427

Characterization of Salmonella ZntR, Zur, and RcnR. β-Galactosidase activity in wild type Salmonella (defined earlier) containing P zntA ( A ) or rcnR- P rcnA ( B ) fused to lacZ following growth to mid-exponential phase in the absence or presence of MNIC MnCl 2 , C 6 H 5 FeO 7 , CoCl 2 , NiSO 4 , CuSO 4 , or ZnSO 4 . C , apo-subtracted UV-visible difference spectra of ZntR (24.9 μ m , monomer) upon titration with CoCl 2 . Inset, binding isotherms at 314 nm ( circles ) and 650 nm ( triangles ). D , apo-subtracted UV-visible difference spectra of Co(II)-ZntR (24.0 μ m , monomer; equilibrated with 1 m eq CoCl 2 ) ( solid line ), and following addition of 0.5 and 1 m eq of ZnCl 2 ( dashed lines ). E , fluorescence emission of ZntR (13.1 μ m , monomer) following titration with ZnCl 2 . F , analysis of fractions (0.5 ml) for protein by Bradford assay ( open circles ) and zinc by ICP-MS ( filled circles ) following size exclusion chromatography of Zur (0.5 ml at 20 μ m , monomer) preincubated with 1 m m EDTA ( left panel ) or 120 μ m ZnCl 2 ( right panel ). G , apo-subtracted UV-visible difference spectra of Zur (24.8 μ m , monomer) upon titration with CoCl 2 . Inset, binding isotherms at 350 nm ( circles ), 576 nm ( triangles ), and 670 nm ( squares ). H , apo-subtracted UV-visible difference spectra of Zur (27.7 μ m , monomer; equilibrated with 2 m eq of CoCl 2 ) ( solid line ) and following titration with ZnCl 2 ( dashed lines ). Inset, quenching of feature at 350 nm. I , representative ( n = 3) mag fura-2 absorbance upon titration of mag fura-2 (12.1 μ m ) with ZnCl 2 in the presence of Zur (11.7 μ m , monomer). Solid line describes competition from Zur for 2 eq of Zn(II) per monomer (four exchangeable sites per dimer, with three independent binding events: K Zn1–2, K Zn3 , and K Zn4 ). Dashed lines are simulated curves with K Zn4 10-fold tighter and 10-fold weaker than fitted K Zn4 ( K Zn1–2 and K Zn3 fixed to fitted values). J , apo-subtracted UV-visible difference spectra of RcnR (30.6 μ m , monomer) upon titration with NiCl 2 . Inset, binding isotherm at 333 nm. K , as J except with RcnR (27.3 μ m , monomer) and CoCl 2 . Inset, binding isotherm at 336 nm. L , apo-subtracted absorbance of RcnR (31.4 μ m , monomer) after addition of 34.5 μ m CoCl 2 and incubation at room temperature under anaerobic conditions in a gas-tight cuvette for 10 min ( solid line ) or 65 h ( dashed line ). Inset, time course at 314 nm.
Figure Legend Snippet: Characterization of Salmonella ZntR, Zur, and RcnR. β-Galactosidase activity in wild type Salmonella (defined earlier) containing P zntA ( A ) or rcnR- P rcnA ( B ) fused to lacZ following growth to mid-exponential phase in the absence or presence of MNIC MnCl 2 , C 6 H 5 FeO 7 , CoCl 2 , NiSO 4 , CuSO 4 , or ZnSO 4 . C , apo-subtracted UV-visible difference spectra of ZntR (24.9 μ m , monomer) upon titration with CoCl 2 . Inset, binding isotherms at 314 nm ( circles ) and 650 nm ( triangles ). D , apo-subtracted UV-visible difference spectra of Co(II)-ZntR (24.0 μ m , monomer; equilibrated with 1 m eq CoCl 2 ) ( solid line ), and following addition of 0.5 and 1 m eq of ZnCl 2 ( dashed lines ). E , fluorescence emission of ZntR (13.1 μ m , monomer) following titration with ZnCl 2 . F , analysis of fractions (0.5 ml) for protein by Bradford assay ( open circles ) and zinc by ICP-MS ( filled circles ) following size exclusion chromatography of Zur (0.5 ml at 20 μ m , monomer) preincubated with 1 m m EDTA ( left panel ) or 120 μ m ZnCl 2 ( right panel ). G , apo-subtracted UV-visible difference spectra of Zur (24.8 μ m , monomer) upon titration with CoCl 2 . Inset, binding isotherms at 350 nm ( circles ), 576 nm ( triangles ), and 670 nm ( squares ). H , apo-subtracted UV-visible difference spectra of Zur (27.7 μ m , monomer; equilibrated with 2 m eq of CoCl 2 ) ( solid line ) and following titration with ZnCl 2 ( dashed lines ). Inset, quenching of feature at 350 nm. I , representative ( n = 3) mag fura-2 absorbance upon titration of mag fura-2 (12.1 μ m ) with ZnCl 2 in the presence of Zur (11.7 μ m , monomer). Solid line describes competition from Zur for 2 eq of Zn(II) per monomer (four exchangeable sites per dimer, with three independent binding events: K Zn1–2, K Zn3 , and K Zn4 ). Dashed lines are simulated curves with K Zn4 10-fold tighter and 10-fold weaker than fitted K Zn4 ( K Zn1–2 and K Zn3 fixed to fitted values). J , apo-subtracted UV-visible difference spectra of RcnR (30.6 μ m , monomer) upon titration with NiCl 2 . Inset, binding isotherm at 333 nm. K , as J except with RcnR (27.3 μ m , monomer) and CoCl 2 . Inset, binding isotherm at 336 nm. L , apo-subtracted absorbance of RcnR (31.4 μ m , monomer) after addition of 34.5 μ m CoCl 2 and incubation at room temperature under anaerobic conditions in a gas-tight cuvette for 10 min ( solid line ) or 65 h ( dashed line ). Inset, time course at 314 nm.

Techniques Used: Activity Assay, Titration, Binding Assay, Fluorescence, Bradford Assay, Mass Spectrometry, Size-exclusion Chromatography, Incubation

13) Product Images from "The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome"

Article Title: The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome

Journal: eLife

doi: 10.7554/eLife.31481

Additional characterization of the H3 tail in the context of the NCP and peptide. ( A ) Plot of the secondary structure for the H3 tail predicted by CSI3.0 as entirely random coil based on chemical shift values. ( B ) 1 H- 15 N HSQC spectra collected on 55 μM 15 N-H3-NCP at 10 (blue), 25 (green), and 37°C (red) at 0 mM added KCl. ( C ) 1 H- 15 N HSQC spectra collected on 55 μM 15 N-H3-NCP at 10 (red), 25 (blue), and 37°C (orange) at 150 mM added KCl. ( D ) 1 H- 15 N HSQC spectra collected on 55 μM 15 N-H3-NCP at 37°C in the presence of increasing concentrations of KCl, color coded according to legend. ( E ) 1 H- 15 N HSQC spectra collected on 55 μM 15 N-H3-NCP at 37°C and 150 mM KCl in the presence of increasing concentrations of MgCl 2 , color coded according to legend. The initial spectrum (0 mM MgCl 2 ) was collected with 2 mM EDTA. ( F ) 1 H- 15 N SOFAST HMQC spectra collected on 110 μM 15 N-H3(1–44) at 10°C. Spectra were collected at increasing concentrations of added KCl, as indicated in the figure. The titration was carried out at 10°C because the peptide signal is significantly attenuated at 25 and especially 37°C. ( G ) Overlay of 1 H- 15 N HSQC/HMQC spectra for 15 N-H3-NCP at 0 mM (green) and 150 mM (gold) added KCl and 15 N-H3(1–44) at 150 mM added KCl (purple). These spectra were all collected at 25°C to bridge the KCl titrations shown in ( D ) and ( F ). There are several confounding factors (e.g. temperature and salt-sensitivity of the free and bound states and differential amide exchange rates) that complicate these spectral overlays, but comparison of these three spectra at 25°C shows a number of residues that are consistent with the idea that KCl can shift the population of collapsed vs. extended tails. An increase in salt concentration is expected to cause an increase in the population of nucleosomal H3 tails that are released (or partially released) from the core DNA. Some of these residues are labeled, with arcs connecting the three states.
Figure Legend Snippet: Additional characterization of the H3 tail in the context of the NCP and peptide. ( A ) Plot of the secondary structure for the H3 tail predicted by CSI3.0 as entirely random coil based on chemical shift values. ( B ) 1 H- 15 N HSQC spectra collected on 55 μM 15 N-H3-NCP at 10 (blue), 25 (green), and 37°C (red) at 0 mM added KCl. ( C ) 1 H- 15 N HSQC spectra collected on 55 μM 15 N-H3-NCP at 10 (red), 25 (blue), and 37°C (orange) at 150 mM added KCl. ( D ) 1 H- 15 N HSQC spectra collected on 55 μM 15 N-H3-NCP at 37°C in the presence of increasing concentrations of KCl, color coded according to legend. ( E ) 1 H- 15 N HSQC spectra collected on 55 μM 15 N-H3-NCP at 37°C and 150 mM KCl in the presence of increasing concentrations of MgCl 2 , color coded according to legend. The initial spectrum (0 mM MgCl 2 ) was collected with 2 mM EDTA. ( F ) 1 H- 15 N SOFAST HMQC spectra collected on 110 μM 15 N-H3(1–44) at 10°C. Spectra were collected at increasing concentrations of added KCl, as indicated in the figure. The titration was carried out at 10°C because the peptide signal is significantly attenuated at 25 and especially 37°C. ( G ) Overlay of 1 H- 15 N HSQC/HMQC spectra for 15 N-H3-NCP at 0 mM (green) and 150 mM (gold) added KCl and 15 N-H3(1–44) at 150 mM added KCl (purple). These spectra were all collected at 25°C to bridge the KCl titrations shown in ( D ) and ( F ). There are several confounding factors (e.g. temperature and salt-sensitivity of the free and bound states and differential amide exchange rates) that complicate these spectral overlays, but comparison of these three spectra at 25°C shows a number of residues that are consistent with the idea that KCl can shift the population of collapsed vs. extended tails. An increase in salt concentration is expected to cause an increase in the population of nucleosomal H3 tails that are released (or partially released) from the core DNA. Some of these residues are labeled, with arcs connecting the three states.

Techniques Used: Titration, Concentration Assay, Labeling

14) Product Images from "gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes"

Article Title: gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes

Journal: The EMBO Journal

doi: 10.1093/emboj/19.7.1458

Fig. 3. gC1q–R is a ligand for InlB. ( A ) N -octyl glucoside extracts were prepared from surface biotin-labeled Vero cell extracts and loaded onto an InlB affinity column. After extensive washing, proteins were eluted with 10 mM EDTA, and 200 μl fractions were collected. Fraction samples (10 μl) were analyzed by SDS–PAGE, transferred onto nitrocellulose and probed with streptavidin coupled to peroxidase to detect biotinylated proteins by chemiluminescence. ( B ). ( C ) Western blot analysis of an elution fraction (15 μl) with streptavidin (1) or with a polyclonal antibody directed against gC1q–R (2). ( D ) Western blot analysis of the cellular expression of gC1q–R. A 100 μg aliquot of solubilized Vero, HEp-2, HeLa or GPC16 cell membrane proteins was separated by SDS–PAGE and transferred onto nitrocellulose. gC1q–R was revealed with an anti-gC1q–R polyclonal antibody.
Figure Legend Snippet: Fig. 3. gC1q–R is a ligand for InlB. ( A ) N -octyl glucoside extracts were prepared from surface biotin-labeled Vero cell extracts and loaded onto an InlB affinity column. After extensive washing, proteins were eluted with 10 mM EDTA, and 200 μl fractions were collected. Fraction samples (10 μl) were analyzed by SDS–PAGE, transferred onto nitrocellulose and probed with streptavidin coupled to peroxidase to detect biotinylated proteins by chemiluminescence. ( B ). ( C ) Western blot analysis of an elution fraction (15 μl) with streptavidin (1) or with a polyclonal antibody directed against gC1q–R (2). ( D ) Western blot analysis of the cellular expression of gC1q–R. A 100 μg aliquot of solubilized Vero, HEp-2, HeLa or GPC16 cell membrane proteins was separated by SDS–PAGE and transferred onto nitrocellulose. gC1q–R was revealed with an anti-gC1q–R polyclonal antibody.

Techniques Used: Labeling, Affinity Column, SDS Page, Western Blot, Expressing

15) Product Images from "Computational design of a water-soluble analog of phospholamban"

Article Title: Computational design of a water-soluble analog of phospholamban

Journal: Protein Science : A Publication of the Protein Society

doi:

CD spectra of 125 μM WSPLB and WSPLB (21–52). WSPLB spectra taken in 10 mM sodium phosphate pH 7.5, 50 mM NaCl, 1 mM TCEP-HCl. WSPLB (21–52) spectra taken in 15 mM MOPS pH 7.0, 50 mM NaCl, 1 mM EDTA, and 1 mM TCEP-HCl.
Figure Legend Snippet: CD spectra of 125 μM WSPLB and WSPLB (21–52). WSPLB spectra taken in 10 mM sodium phosphate pH 7.5, 50 mM NaCl, 1 mM TCEP-HCl. WSPLB (21–52) spectra taken in 15 mM MOPS pH 7.0, 50 mM NaCl, 1 mM EDTA, and 1 mM TCEP-HCl.

Techniques Used:

Sedimentation equilibrium of WSPLB in 100 mM NaCl, 25 mM MOPS pH 7.5, 1 mM EDTA, 1 mM TCEP-HCl at 35,000 rpm. Equilibrium A 280 -radius profiles for three cell compartments containing peptide concentrations of 15, 39, and 113 μM are shown. ( A ) Shows the data fit to a monomer–pentamer equilibrium, while ( B ) shows a less satisfactory data fit to a monomer–tetramer equilibrium.
Figure Legend Snippet: Sedimentation equilibrium of WSPLB in 100 mM NaCl, 25 mM MOPS pH 7.5, 1 mM EDTA, 1 mM TCEP-HCl at 35,000 rpm. Equilibrium A 280 -radius profiles for three cell compartments containing peptide concentrations of 15, 39, and 113 μM are shown. ( A ) Shows the data fit to a monomer–pentamer equilibrium, while ( B ) shows a less satisfactory data fit to a monomer–tetramer equilibrium.

Techniques Used: Sedimentation

16) Product Images from "The Tomato R Gene Products I-2 and Mi-1 Are Functional ATP Binding Proteins with ATPase Activity"

Article Title: The Tomato R Gene Products I-2 and Mi-1 Are Functional ATP Binding Proteins with ATPase Activity

Journal: The Plant Cell

doi: 10.1105/tpc.005793

Influence of Ion Concentrations and pH on ATP Binding of I-2N. α- 32 P-ATP binding was measured in a filter binding assay as described in Methods. (A) to (C) The maximal amount of α- 32 P-ATP bound was set to 1. (A) I-2N (1 μM) was incubated with 0.07 μM α- 32 P-ATP (6.3 × 10 5 cpm/pmol) in the standard reaction mixture except for a variable NaCl concentration. (B) I-2N (1.5 μM) was incubated with 0.1 μM α- 32 P-ATP (4.3 × 10 5 cpm/pmol) in the standard reaction mixture except for a variable pH. (C) I-2N (0.8 μM) was incubated with 0.2 μM α- 32 P-ATP (2.1 × 10 5 cpm/pmol) in the standard reaction mixture except for a variable MgCl 2 concentration and the absence of DTT and EDTA. (D) I-2N (0.8 μM) was incubated with 0.2 μM α- 32 P-ATP (2.1 × 10 5 cpm/pmol) in the standard reaction mixture except for variable concentrations of the divalent (Div.) cations of MgCl 2 , MnCl 2 , or CaCl 2 . The maximal amount of α- 32 P-ATP bound in presence of MgCl 2 was set to 1.
Figure Legend Snippet: Influence of Ion Concentrations and pH on ATP Binding of I-2N. α- 32 P-ATP binding was measured in a filter binding assay as described in Methods. (A) to (C) The maximal amount of α- 32 P-ATP bound was set to 1. (A) I-2N (1 μM) was incubated with 0.07 μM α- 32 P-ATP (6.3 × 10 5 cpm/pmol) in the standard reaction mixture except for a variable NaCl concentration. (B) I-2N (1.5 μM) was incubated with 0.1 μM α- 32 P-ATP (4.3 × 10 5 cpm/pmol) in the standard reaction mixture except for a variable pH. (C) I-2N (0.8 μM) was incubated with 0.2 μM α- 32 P-ATP (2.1 × 10 5 cpm/pmol) in the standard reaction mixture except for a variable MgCl 2 concentration and the absence of DTT and EDTA. (D) I-2N (0.8 μM) was incubated with 0.2 μM α- 32 P-ATP (2.1 × 10 5 cpm/pmol) in the standard reaction mixture except for variable concentrations of the divalent (Div.) cations of MgCl 2 , MnCl 2 , or CaCl 2 . The maximal amount of α- 32 P-ATP bound in presence of MgCl 2 was set to 1.

Techniques Used: Binding Assay, Filter-binding Assay, Incubation, Concentration Assay

17) Product Images from "Analysis of Peptides and Proteins in Their Binding to GroEL"

Article Title: Analysis of Peptides and Proteins in Their Binding to GroEL

Journal: Journal of peptide science : an official publication of the European Peptide Society

doi: 10.1002/psc.1288

ITC data at 20 °C and the temperature dependence of enthalpic change (insert) of: A) SBP association with GroEL; B) SBP association with GroEL/GroES; C) α-lactalbumin association with GroEL; D) α-lactalbumin association with GroEL/GroES. Shown are the integrations of heat exchange (after background correction) for each injection during titration, and the line represents the fit to a single-site binding model. A), 21.4 μM GroEL was titrated into 30 μM SBP in 50 mM TrisCl, pH 7.5, 150 mM NaCl, and 1 mM EDTA; B), 21.4 μM GroEL/GroES was titrated into 30 μM SBP in 50 mM TrisCl, pH 7.5, 10 mM MgCl 2 , 200 mM KCl, 1 mM EDTA 0.05% NaN 3 , 5 mM DTT, and 50 μM ADP; C), 50 μM α-lactalbumin was titrated into 21.4 μM GroEL in 50 mM Hepes, pH 7.2, 200 mM KCl, 5 mM DTT and 1 mM EDTA; D), 50 μM α-lactalbumin was titrated into 21.4 μM GroEL/GroES in 50 mM TrisCl, pH 7.5, 10 mM MgCl 2 , 200 mM KCl, 1 mM EDTA 0.05% NaN 3 , 5 mM DTT, and 50 μM ADP.
Figure Legend Snippet: ITC data at 20 °C and the temperature dependence of enthalpic change (insert) of: A) SBP association with GroEL; B) SBP association with GroEL/GroES; C) α-lactalbumin association with GroEL; D) α-lactalbumin association with GroEL/GroES. Shown are the integrations of heat exchange (after background correction) for each injection during titration, and the line represents the fit to a single-site binding model. A), 21.4 μM GroEL was titrated into 30 μM SBP in 50 mM TrisCl, pH 7.5, 150 mM NaCl, and 1 mM EDTA; B), 21.4 μM GroEL/GroES was titrated into 30 μM SBP in 50 mM TrisCl, pH 7.5, 10 mM MgCl 2 , 200 mM KCl, 1 mM EDTA 0.05% NaN 3 , 5 mM DTT, and 50 μM ADP; C), 50 μM α-lactalbumin was titrated into 21.4 μM GroEL in 50 mM Hepes, pH 7.2, 200 mM KCl, 5 mM DTT and 1 mM EDTA; D), 50 μM α-lactalbumin was titrated into 21.4 μM GroEL/GroES in 50 mM TrisCl, pH 7.5, 10 mM MgCl 2 , 200 mM KCl, 1 mM EDTA 0.05% NaN 3 , 5 mM DTT, and 50 μM ADP.

Techniques Used: Injection, Titration, Binding Assay

18) Product Images from "The electron distribution in the “activated” state of cytochrome c oxidase"

Article Title: The electron distribution in the “activated” state of cytochrome c oxidase

Journal: Scientific Reports

doi: 10.1038/s41598-018-25779-w

Absorbance difference spectra. A comparison of reduced minus oxidized difference spectra of the Cyt c O-cyt. c complex (black) and the sum of the reduced minus oxidized difference spectra of Cyt c O and cyt. c (red), respectively. Spectra of the oxidized states were recorded first and then the atmosphere in the cuvette was replaced for N 2 after which the samples were reduced with ascorbate (2 mM) and hexaammineruthenium(II) chloride (1 μM). Reduction of the samples was followed in time by recording spectra over ~2 hours until no further changes were observed. The data to the right of the axis break have been multiplied by a factor of five. The concentrations of Cyt c O and cyt. c were ~3 μM and ~5 μM, respectively, in 50 mM HEPES (pH 7.5), 0.05% DDM and 100 µM EDTA.
Figure Legend Snippet: Absorbance difference spectra. A comparison of reduced minus oxidized difference spectra of the Cyt c O-cyt. c complex (black) and the sum of the reduced minus oxidized difference spectra of Cyt c O and cyt. c (red), respectively. Spectra of the oxidized states were recorded first and then the atmosphere in the cuvette was replaced for N 2 after which the samples were reduced with ascorbate (2 mM) and hexaammineruthenium(II) chloride (1 μM). Reduction of the samples was followed in time by recording spectra over ~2 hours until no further changes were observed. The data to the right of the axis break have been multiplied by a factor of five. The concentrations of Cyt c O and cyt. c were ~3 μM and ~5 μM, respectively, in 50 mM HEPES (pH 7.5), 0.05% DDM and 100 µM EDTA.

Techniques Used:

19) Product Images from "Polyadenylation inhibition by the triphosphates of deoxyadenosine analogues"

Article Title: Polyadenylation inhibition by the triphosphates of deoxyadenosine analogues

Journal: Leukemia research

doi: 10.1016/j.leukres.2008.03.010

Substrate specificity of yPAP toward various purine triphosphate analogues (A) Elongation of RNA primer 5′ UGU GCC CGA 3′ by yPAP. A mixture containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4 U/μL yPAP, 250 μM analogue triphosphate, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled unextended primer (no NTP); lane 3 , ATP; lane 4 , 2′-dATP; lane 5 , 3′-dATP; lane 6 , 2-Cl-ATP; lane 7 , 2-Cl-dATP; lane 8 , ara-ATP; lane 9 , F-ara-ATP; lane 10 , Cl-F-ara-ATP; lane 11 , Cl-F-dATP, lane 12 , GTP; lane 13 , dGTP, lane 14 , ara-GTP. (B) Graphical representation of RNA primer extension by yPAP with various modified triphosphates. Shown is the distribution of single extension products (white) and full extension products beyond first incorporation (hatched) as a percentage of the total counts in each lane, as determined using ImageQuant software. These experiments were conducted in triplicate with similar results.
Figure Legend Snippet: Substrate specificity of yPAP toward various purine triphosphate analogues (A) Elongation of RNA primer 5′ UGU GCC CGA 3′ by yPAP. A mixture containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4 U/μL yPAP, 250 μM analogue triphosphate, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled unextended primer (no NTP); lane 3 , ATP; lane 4 , 2′-dATP; lane 5 , 3′-dATP; lane 6 , 2-Cl-ATP; lane 7 , 2-Cl-dATP; lane 8 , ara-ATP; lane 9 , F-ara-ATP; lane 10 , Cl-F-ara-ATP; lane 11 , Cl-F-dATP, lane 12 , GTP; lane 13 , dGTP, lane 14 , ara-GTP. (B) Graphical representation of RNA primer extension by yPAP with various modified triphosphates. Shown is the distribution of single extension products (white) and full extension products beyond first incorporation (hatched) as a percentage of the total counts in each lane, as determined using ImageQuant software. These experiments were conducted in triplicate with similar results.

Techniques Used: Incubation, Acetylene Reduction Assay, Modification, Software

Reduction of poly(A) tail length by modified ATP analogues Gel electrophoretic analyses of an RNA primer 5′ UGU GCC CGA 3′ with yPAP, 250 μM ATP, and increasing amounts of ATP analogue: (A) F-ara-ATP, (B) Ara-ATP, (C) 2-Cl-dATP, (D) Cl-F-ara-ATP and (E) Cl-F-dATP. For each set of reactions, a solution (5 μL) containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4U/μL yPAP, 100 μM ATP, 0–250 μM analogue ATP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/mL acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled primer (no triphosphates); lane 3 , ATP only; lanes 4–14 , 250 μM ATP with 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 μM analogue triphosphate, respectively. (F) Graphical representation of figures (A–E). The extension of poly(A)-tail length for the varying concentrations of F-ara-ATP (■), Ara-ATP (▲), 2-Cl-dATP (◆), Cl-F-ara-ATP (▼), and Cl-F-dATP (●) were measured using ImageQuant analysis and then compared with control (without analogue) RNA primer extensions and expressed as a percentage of control. These experiments were conducted in triplicate with similar results.
Figure Legend Snippet: Reduction of poly(A) tail length by modified ATP analogues Gel electrophoretic analyses of an RNA primer 5′ UGU GCC CGA 3′ with yPAP, 250 μM ATP, and increasing amounts of ATP analogue: (A) F-ara-ATP, (B) Ara-ATP, (C) 2-Cl-dATP, (D) Cl-F-ara-ATP and (E) Cl-F-dATP. For each set of reactions, a solution (5 μL) containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4U/μL yPAP, 100 μM ATP, 0–250 μM analogue ATP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/mL acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled primer (no triphosphates); lane 3 , ATP only; lanes 4–14 , 250 μM ATP with 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 μM analogue triphosphate, respectively. (F) Graphical representation of figures (A–E). The extension of poly(A)-tail length for the varying concentrations of F-ara-ATP (■), Ara-ATP (▲), 2-Cl-dATP (◆), Cl-F-ara-ATP (▼), and Cl-F-dATP (●) were measured using ImageQuant analysis and then compared with control (without analogue) RNA primer extensions and expressed as a percentage of control. These experiments were conducted in triplicate with similar results.

Techniques Used: Modification, Acetylene Reduction Assay, Incubation

20) Product Images from "A Viral Histone H4 Joins to Eukaryotic Nucleosomes and Alters Host Gene Expression"

Article Title: A Viral Histone H4 Joins to Eukaryotic Nucleosomes and Alters Host Gene Expression

Journal: Journal of Virology

doi: 10.1128/JVI.01759-13

In vitro reconstruction of a viral histone H4, CpBV-H4, with eukaryotic nucleosome components. (A) Size exclusion chromatography. Two milligrams of nucleosomal components of unfolded Xenopus laevis H2A, H2B, H3, and H4 were mixed with unfolded CpBV-H4 in an equal concentration and refolded by dialysis against a refolding buffer (20 mM Tris [pH 7.5], 2 M NaCl, 2 mM β-mercaptoethanol, 0.5 mM EDTA) overnight. The resulting complex was then analyzed by using passage through a Supradex S20 column and monitoring absorbance at 280 nm as a function of elution volume. In the size exclusion chromatogram, two main peaks corresponded to octamer and tetramer reference. mAU, milliabsorbance units. (B) The purified fraction was analyzed by 15% SDS-PAGE. Numbers 11 to 18 represent fraction numbers of the size exclusion chromatography. M, molecular mass markers; CA, component analysis of five nucleosomal components used in the in vitro reconstitution assay.
Figure Legend Snippet: In vitro reconstruction of a viral histone H4, CpBV-H4, with eukaryotic nucleosome components. (A) Size exclusion chromatography. Two milligrams of nucleosomal components of unfolded Xenopus laevis H2A, H2B, H3, and H4 were mixed with unfolded CpBV-H4 in an equal concentration and refolded by dialysis against a refolding buffer (20 mM Tris [pH 7.5], 2 M NaCl, 2 mM β-mercaptoethanol, 0.5 mM EDTA) overnight. The resulting complex was then analyzed by using passage through a Supradex S20 column and monitoring absorbance at 280 nm as a function of elution volume. In the size exclusion chromatogram, two main peaks corresponded to octamer and tetramer reference. mAU, milliabsorbance units. (B) The purified fraction was analyzed by 15% SDS-PAGE. Numbers 11 to 18 represent fraction numbers of the size exclusion chromatography. M, molecular mass markers; CA, component analysis of five nucleosomal components used in the in vitro reconstitution assay.

Techniques Used: In Vitro, Size-exclusion Chromatography, Concentration Assay, Purification, SDS Page, Reconstitution Assay

21) Product Images from "A [3Fe-4S] cluster is required for tRNA thiolation in archaea and eukaryotes"

Article Title: A [3Fe-4S] cluster is required for tRNA thiolation in archaea and eukaryotes

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1615732113

The [3Fe-4S] cluster in M. maripaludis ThiI is essential for the tRNA thiolation activity. ( A ) The tRNA thiolation activity of the as-purified ( Left ), EDTA-treated ( Center ), and in vitro reconstituted M. maripaludis ThiI ( Right ) was analyzed with the
Figure Legend Snippet: The [3Fe-4S] cluster in M. maripaludis ThiI is essential for the tRNA thiolation activity. ( A ) The tRNA thiolation activity of the as-purified ( Left ), EDTA-treated ( Center ), and in vitro reconstituted M. maripaludis ThiI ( Right ) was analyzed with the

Techniques Used: Activity Assay, Purification, In Vitro

22) Product Images from "Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris"

Article Title: Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris

Journal: Applied Microbiology and Biotechnology

doi: 10.1007/s00253-013-5264-4

Purification of rhIL-25 protein. a The fermentation supernatant was concentrated by ultrafiltration and then the buffer exchanged against 50 mM Tris–HCl and 1 mM EDTA, loaded onto Q sepharose Fast Flow resin, and eluted using NaCl gradient from 0 to 1 M. The rhIL-25 protein was flowing through the Q Sepharose FF, while the containments were bound by the column. b The fractions were separated on 15 % SDS-PAGE. Lane M molecular weight marker. Lane 1 fermentation supernatant at 44 h after methanol induction. Lane 2 after buffer exchange. Lane 3 flow-through of Q FF. Lane 4 wash-off using balance buffer. Lane 5 – 9 eluate from Q Sepharose FF
Figure Legend Snippet: Purification of rhIL-25 protein. a The fermentation supernatant was concentrated by ultrafiltration and then the buffer exchanged against 50 mM Tris–HCl and 1 mM EDTA, loaded onto Q sepharose Fast Flow resin, and eluted using NaCl gradient from 0 to 1 M. The rhIL-25 protein was flowing through the Q Sepharose FF, while the containments were bound by the column. b The fractions were separated on 15 % SDS-PAGE. Lane M molecular weight marker. Lane 1 fermentation supernatant at 44 h after methanol induction. Lane 2 after buffer exchange. Lane 3 flow-through of Q FF. Lane 4 wash-off using balance buffer. Lane 5 – 9 eluate from Q Sepharose FF

Techniques Used: Purification, Flow Cytometry, SDS Page, Molecular Weight, Marker, Buffer Exchange

23) Product Images from "EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus, et al. EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus"

Article Title: EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus, et al. EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13962

Study of the presence of C1s in IC : (A and B), 50 μl volumes of plasma samples from healthy controls and SLE patients were incubated with protein‐G‐Sepharose to precipitate the IgG and IgG‐ IC . The bound fractions were analyzed by gelatin zymography in the absence (A) or presence (B) of 10 m mol L −1 EDTA . C, Western blot analysis of the same fractions shown in panels A and B for the detection of C1s. D, Quantification of the bands at 90‐80 kDa detected in the zymography from panel B. E, Quantification of the Western blot analysis. P values were determined by ANOVA Kruskal‐Wallis test, * P
Figure Legend Snippet: Study of the presence of C1s in IC : (A and B), 50 μl volumes of plasma samples from healthy controls and SLE patients were incubated with protein‐G‐Sepharose to precipitate the IgG and IgG‐ IC . The bound fractions were analyzed by gelatin zymography in the absence (A) or presence (B) of 10 m mol L −1 EDTA . C, Western blot analysis of the same fractions shown in panels A and B for the detection of C1s. D, Quantification of the bands at 90‐80 kDa detected in the zymography from panel B. E, Quantification of the Western blot analysis. P values were determined by ANOVA Kruskal‐Wallis test, * P

Techniques Used: Incubation, Zymography, Western Blot

Identification of an unknown protease with higher levels in SLE versus control plasma: A, 2 μl of plasma sample from healthy controls (C4‐C9) and SLE patients (P3‐P7) was analyzed by gelatin zymography. B, Analysis of similar aliquots as in panel A in the presence of 10 m mol L −1 EDTA. C, 50 μl of plasma samples from SLE patients and healthy controls were incubated with gelatin‐Sepharose. The unbound (UB) and bound (B) fractions were analyzed by gelatin zymography. D, Quantification of the unknown‐protein bands by gelatin zymography analysis in the presence of 10 m mol L −1 EDTA (panel B). P values were determined by ANOVA Kruskal‐Wallis test, *** P
Figure Legend Snippet: Identification of an unknown protease with higher levels in SLE versus control plasma: A, 2 μl of plasma sample from healthy controls (C4‐C9) and SLE patients (P3‐P7) was analyzed by gelatin zymography. B, Analysis of similar aliquots as in panel A in the presence of 10 m mol L −1 EDTA. C, 50 μl of plasma samples from SLE patients and healthy controls were incubated with gelatin‐Sepharose. The unbound (UB) and bound (B) fractions were analyzed by gelatin zymography. D, Quantification of the unknown‐protein bands by gelatin zymography analysis in the presence of 10 m mol L −1 EDTA (panel B). P values were determined by ANOVA Kruskal‐Wallis test, *** P

Techniques Used: Zymography, Incubation

24) Product Images from "EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus, et al. EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus"

Article Title: EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus, et al. EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13962

Identification of an unknown protease with higher levels in SLE versus control plasma: A, 2 μl of plasma sample from healthy controls (C4‐C9) and SLE patients (P3‐P7) was analyzed by gelatin zymography. B, Analysis of similar aliquots as in panel A in the presence of 10 m mol L −1 EDTA. C, 50 μl of plasma samples from SLE patients and healthy controls were incubated with gelatin‐Sepharose. The unbound (UB) and bound (B) fractions were analyzed by gelatin zymography. D, Quantification of the unknown‐protein bands by gelatin zymography analysis in the presence of 10 m mol L −1 EDTA (panel B). P values were determined by ANOVA Kruskal‐Wallis test, *** P
Figure Legend Snippet: Identification of an unknown protease with higher levels in SLE versus control plasma: A, 2 μl of plasma sample from healthy controls (C4‐C9) and SLE patients (P3‐P7) was analyzed by gelatin zymography. B, Analysis of similar aliquots as in panel A in the presence of 10 m mol L −1 EDTA. C, 50 μl of plasma samples from SLE patients and healthy controls were incubated with gelatin‐Sepharose. The unbound (UB) and bound (B) fractions were analyzed by gelatin zymography. D, Quantification of the unknown‐protein bands by gelatin zymography analysis in the presence of 10 m mol L −1 EDTA (panel B). P values were determined by ANOVA Kruskal‐Wallis test, *** P

Techniques Used: Zymography, Incubation

25) Product Images from "Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A"

Article Title: Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp157

Human mtTFA is an asymmetric monomer in the absence of DNA. ( A ) Deletion constructs of h-mtTFA. The upper panel shows a schematic diagram and the lower panel shows SDS–PAGE of h-mtTFA deletion constructs on a 15% polyacrylamide gel. ( B ) Size-exclusion chromatography (Superdex 200; GE Healthcare) elution profiles of h-mtTFA and h-mtTFA deletion constructs, mtTFA 1–179 , mtTFA 1–109 , mtTFA 1–79 , mtTFA 80–204 , mtTFA 110–204 and mtTFA 110–179 (top panel), and the single HMG domains, HMGB1 box A and HMGD (lower panel) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The position of each size standard is indicated by arrows above the top panel for amylase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and RNase A (14 kDa). The void volume was at 45 ml and is not shown. ( C ) Sedimentation velocity profiles for the raw data acquired at different time points and the residuals after fittings had been performed using SEDFIT in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. ( D ) Calculated sedimentation coefficient distributions for the full-length h-mtTFA.
Figure Legend Snippet: Human mtTFA is an asymmetric monomer in the absence of DNA. ( A ) Deletion constructs of h-mtTFA. The upper panel shows a schematic diagram and the lower panel shows SDS–PAGE of h-mtTFA deletion constructs on a 15% polyacrylamide gel. ( B ) Size-exclusion chromatography (Superdex 200; GE Healthcare) elution profiles of h-mtTFA and h-mtTFA deletion constructs, mtTFA 1–179 , mtTFA 1–109 , mtTFA 1–79 , mtTFA 80–204 , mtTFA 110–204 and mtTFA 110–179 (top panel), and the single HMG domains, HMGB1 box A and HMGD (lower panel) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The position of each size standard is indicated by arrows above the top panel for amylase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and RNase A (14 kDa). The void volume was at 45 ml and is not shown. ( C ) Sedimentation velocity profiles for the raw data acquired at different time points and the residuals after fittings had been performed using SEDFIT in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. ( D ) Calculated sedimentation coefficient distributions for the full-length h-mtTFA.

Techniques Used: Construct, SDS Page, Size-exclusion Chromatography, Sedimentation

Human mtTFA box B interacts with other regions of mtTFA. An N-terminal GST fusion with box B (mtTFA 110–179 ) was tested for its ability to interact with the various deletion constructs of h-mtTFA (mtTFA 1-109 , mtTFA 1–79 , mtTFA 1–80–204 , mtTFA 110–204 , mtTFA 110–179 , mtTFA 80–179 , mtTFA 1–95 and mtTFA 96–179 ) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The reactions were electrophoresed on a 15% SDS–PAGE gel and transferred to nitrocellulose membrane (Millipore) and probed with anti-His and anti-GST antibodies, respectively.
Figure Legend Snippet: Human mtTFA box B interacts with other regions of mtTFA. An N-terminal GST fusion with box B (mtTFA 110–179 ) was tested for its ability to interact with the various deletion constructs of h-mtTFA (mtTFA 1-109 , mtTFA 1–79 , mtTFA 1–80–204 , mtTFA 110–204 , mtTFA 110–179 , mtTFA 80–179 , mtTFA 1–95 and mtTFA 96–179 ) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The reactions were electrophoresed on a 15% SDS–PAGE gel and transferred to nitrocellulose membrane (Millipore) and probed with anti-His and anti-GST antibodies, respectively.

Techniques Used: Construct, SDS Page

26) Product Images from "1H, 13C, and 15N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin ligase ITCH"

Article Title: 1H, 13C, and 15N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin ligase ITCH

Journal: Biomolecular Nmr Assignments

doi: 10.1007/s12104-018-9843-2

Assigned observable 1 H– 15 N HSQC spectrum of the human HECT C -terminal lobe of ITCH (residues 784–903, 2.6 mM). The spectrum is labeled according to the one-amino acid code and residue number of the human ITCH sequence. The NMR sample contained 13 C and 15 N-isotopically enriched ITCH in 20 mM MES pH 6.0, 120 mM NaCl, 1 mM EDTA, and 10% D 2 O. Data was collected on a Varian Inova 600-MHz NMR spectrometer at 25 °C. Peaks corresponding to asparagine and glutamine side chain amides are connected with a horizontal line
Figure Legend Snippet: Assigned observable 1 H– 15 N HSQC spectrum of the human HECT C -terminal lobe of ITCH (residues 784–903, 2.6 mM). The spectrum is labeled according to the one-amino acid code and residue number of the human ITCH sequence. The NMR sample contained 13 C and 15 N-isotopically enriched ITCH in 20 mM MES pH 6.0, 120 mM NaCl, 1 mM EDTA, and 10% D 2 O. Data was collected on a Varian Inova 600-MHz NMR spectrometer at 25 °C. Peaks corresponding to asparagine and glutamine side chain amides are connected with a horizontal line

Techniques Used: Labeling, Sequencing, Nuclear Magnetic Resonance

27) Product Images from "A Unique Arabinose 5-Phosphate Isomerase Found within a Genomic Island Associated with the Uropathogenicity of Escherichia coli CFT073 ▿"

Article Title: A Unique Arabinose 5-Phosphate Isomerase Found within a Genomic Island Associated with the Uropathogenicity of Escherichia coli CFT073 ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00033-11

Effects of divalent metals on the activity of c3406. The enzyme was incubated with no additive (As Isolated), EDTA, or a divalent metal ion as described in Materials and Methods and then assayed for activity. The activity of the EDTA-treated sample was
Figure Legend Snippet: Effects of divalent metals on the activity of c3406. The enzyme was incubated with no additive (As Isolated), EDTA, or a divalent metal ion as described in Materials and Methods and then assayed for activity. The activity of the EDTA-treated sample was

Techniques Used: Activity Assay, Incubation, Isolation

28) Product Images from "Identification of Human S100A9 as a Novel Target for Treatment of Autoimmune Disease via Binding to Quinoline-3-Carboxamides"

Article Title: Identification of Human S100A9 as a Novel Target for Treatment of Autoimmune Disease via Binding to Quinoline-3-Carboxamides

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1000097

Human S100A9 Is a Target Protein for Quinolines (A) The basic structure of the quinoline compounds is shown. In the lower part of the panel, the specific modifications made in order to use these compounds as probes to isolate the target protein are shown. (B) A two-dimensional gel is shown in which the indicated spots (boxed) were subsequently identified as S100A9. The protein in all three spots was isolated separately and homogenously identified as S100A9. (C) Sensorgrams obtained after injection of 25–200 nM human S100A9 over immobilised ABR-224649 (left panel). Sensorgrams from top to bottom represent: 200, 150, 100, 75, 50, 37.5, and 25 nM S100A9 and nonspecific binding (NSB), i.e., sample buffer without S100A9. In this particular experiment, injection time was 6 min at a flow rate of 30 μl/min, and regeneration was performed with a 30-μl pulse of HBS-P buffer containing 3 mM EDTA (HBS-EP). Start injection of sample: association phase (1), running buffer: dissociation phase (2), regeneration solution (3), and running buffer again (4) constitute an analysis cycle. HBS-P with 1 mM Ca 2+ and 10 μM Zn 2+ was used as running and sample buffer. In the right panel, responses at steady state (after subtraction of signal in reference flow cell) were plotted versus concentration of S100A9 yielding half-maximal binding at 85 nM ( r 2 = 1.00). (D) Binding of homo- and heterodimeric human S100A8 and S100A9 to immobilized ABR-224649 at a concentration of 100 nM (based on their homo- or heterodimeric molecular weight). The response at late association phase was calculated and plotted in ascending order of response magnitude. (E) Displacement of S100A9 binding to immobilised ABR-224649 by ABR-215757 is shown. S100A9 was injected for 3 min at 100 nM (i.e., at ≈ B max /2 concentration) ± 7.81–1,000 μM 215757, and responses at late association were plotted against the concentration of competitor. An IC 50 value of 37 μM was calculated in this experiment using a one-site competition model ( r 2 = 1.00). The amino-linker compound, ABR-224649, showed a very similar ability to displace binding as ABR-215757 when coinjected with S100A9 over the surface (unpublished data). (F) Effect of Ca 2+ and Zn 2+ on binding of S100A9 to ABR-224649 is shown. S100A9, 100 nM, was injected in HBS-P buffer containing either a fixed concentration of Ca 2+ (1 mM) or Zn 2+ (10 μM), with Zn 2+ and Ca 2+ concentrations titrated from 0–50 μM and 0–2,000 μM, respectively. Responses at late association phase were plotted versus metal ion concentration, and EC 50 values of 5.5 μM for Zn 2+ and 193 μM for Ca 2+ were calculated using a sigmoidal dose-response model.
Figure Legend Snippet: Human S100A9 Is a Target Protein for Quinolines (A) The basic structure of the quinoline compounds is shown. In the lower part of the panel, the specific modifications made in order to use these compounds as probes to isolate the target protein are shown. (B) A two-dimensional gel is shown in which the indicated spots (boxed) were subsequently identified as S100A9. The protein in all three spots was isolated separately and homogenously identified as S100A9. (C) Sensorgrams obtained after injection of 25–200 nM human S100A9 over immobilised ABR-224649 (left panel). Sensorgrams from top to bottom represent: 200, 150, 100, 75, 50, 37.5, and 25 nM S100A9 and nonspecific binding (NSB), i.e., sample buffer without S100A9. In this particular experiment, injection time was 6 min at a flow rate of 30 μl/min, and regeneration was performed with a 30-μl pulse of HBS-P buffer containing 3 mM EDTA (HBS-EP). Start injection of sample: association phase (1), running buffer: dissociation phase (2), regeneration solution (3), and running buffer again (4) constitute an analysis cycle. HBS-P with 1 mM Ca 2+ and 10 μM Zn 2+ was used as running and sample buffer. In the right panel, responses at steady state (after subtraction of signal in reference flow cell) were plotted versus concentration of S100A9 yielding half-maximal binding at 85 nM ( r 2 = 1.00). (D) Binding of homo- and heterodimeric human S100A8 and S100A9 to immobilized ABR-224649 at a concentration of 100 nM (based on their homo- or heterodimeric molecular weight). The response at late association phase was calculated and plotted in ascending order of response magnitude. (E) Displacement of S100A9 binding to immobilised ABR-224649 by ABR-215757 is shown. S100A9 was injected for 3 min at 100 nM (i.e., at ≈ B max /2 concentration) ± 7.81–1,000 μM 215757, and responses at late association were plotted against the concentration of competitor. An IC 50 value of 37 μM was calculated in this experiment using a one-site competition model ( r 2 = 1.00). The amino-linker compound, ABR-224649, showed a very similar ability to displace binding as ABR-215757 when coinjected with S100A9 over the surface (unpublished data). (F) Effect of Ca 2+ and Zn 2+ on binding of S100A9 to ABR-224649 is shown. S100A9, 100 nM, was injected in HBS-P buffer containing either a fixed concentration of Ca 2+ (1 mM) or Zn 2+ (10 μM), with Zn 2+ and Ca 2+ concentrations titrated from 0–50 μM and 0–2,000 μM, respectively. Responses at late association phase were plotted versus metal ion concentration, and EC 50 values of 5.5 μM for Zn 2+ and 193 μM for Ca 2+ were calculated using a sigmoidal dose-response model.

Techniques Used: Two-Dimensional Gel Electrophoresis, Isolation, Injection, Binding Assay, Flow Cytometry, Concentration Assay, Molecular Weight

29) Product Images from "The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity"

Article Title: The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0080967

Substrate specificity of RM.BpuSI MTase activity. (A) In vitro MTase activity. Indicated oligonucleotide duplexes were incubated with 8-fold molar excess of purified WT or N406A RM.BpuSI or M1.BpuSI in the presence of 0.5 μM 3 H-SAM and 5 mM EDTA or MgCl 2 at 37°C for 1 h. Radioactivity incorporation was measured by scintillation counting filter-bound DNA.. Values shown are average of triplicate experiments and subtracted from background readings without enzyme added. Error bars represent standard errors from the triplicates. (B) Unmodified but not M2-modified sub01 were cleaved into expected products in the presence of Mg 2+ in the MTase activity assays.
Figure Legend Snippet: Substrate specificity of RM.BpuSI MTase activity. (A) In vitro MTase activity. Indicated oligonucleotide duplexes were incubated with 8-fold molar excess of purified WT or N406A RM.BpuSI or M1.BpuSI in the presence of 0.5 μM 3 H-SAM and 5 mM EDTA or MgCl 2 at 37°C for 1 h. Radioactivity incorporation was measured by scintillation counting filter-bound DNA.. Values shown are average of triplicate experiments and subtracted from background readings without enzyme added. Error bars represent standard errors from the triplicates. (B) Unmodified but not M2-modified sub01 were cleaved into expected products in the presence of Mg 2+ in the MTase activity assays.

Techniques Used: Activity Assay, In Vitro, Incubation, Purification, Radioactivity, Modification

30) Product Images from "Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities"

Article Title: Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191819

Steady-state kinetic parameters for mAOX1-4 with aromatic aldehydes as substrates containing a benzyl-group. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Figure Legend Snippet: Steady-state kinetic parameters for mAOX1-4 with aromatic aldehydes as substrates containing a benzyl-group. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

Techniques Used: Variant Assay, Activity Assay

Steady-state kinetic parameters for mAOX1-4 with N-heterocyclic compounds as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Figure Legend Snippet: Steady-state kinetic parameters for mAOX1-4 with N-heterocyclic compounds as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

Techniques Used: Variant Assay, Activity Assay

Steady-state kinetic parameters for mAOX1-4 with aliphatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Figure Legend Snippet: Steady-state kinetic parameters for mAOX1-4 with aliphatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

Techniques Used: Variant Assay, Activity Assay

Steady-state kinetic parameters for mAOX1-4 with cinnamaldehyde-related compounds as substrates aromatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Figure Legend Snippet: Steady-state kinetic parameters for mAOX1-4 with cinnamaldehyde-related compounds as substrates aromatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

Techniques Used: Variant Assay, Activity Assay

31) Product Images from "Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU"

Article Title: Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU

Journal: PLoS ONE

doi: 10.1371/journal.pone.0144510

Q2RNA and its DNA counterpart adopt G4 structures. (A) Far-UV CD spectra of Q2RNA and Q2DNA obtained in 10 mM Tris, pH 7.5, 100 mM KCl, 1 mM EDTA buffer of Q2RNA at 20°C (closed circles) and at 80°C (open circles) as well as of Q2DNA at 20°C (closed triangles) and at 80°C (closed squares). (B) CD melting curves of Q2RNA ( —— ) and Q2DNA ( ------ ) monitored by spectropolarimetry at 264 nm in the same buffer.
Figure Legend Snippet: Q2RNA and its DNA counterpart adopt G4 structures. (A) Far-UV CD spectra of Q2RNA and Q2DNA obtained in 10 mM Tris, pH 7.5, 100 mM KCl, 1 mM EDTA buffer of Q2RNA at 20°C (closed circles) and at 80°C (open circles) as well as of Q2DNA at 20°C (closed triangles) and at 80°C (closed squares). (B) CD melting curves of Q2RNA ( —— ) and Q2DNA ( ------ ) monitored by spectropolarimetry at 264 nm in the same buffer.

Techniques Used:

Q2RNA, but not Q2DNA, stains with a parallel G4 dye. (A) Schematic showing the G4 forming regions of PITX1 mRNA; the RNA and DNA equivalent sequences with the guanylate tracts underlined are also shown. 250 pmol of Q2RNA and Q2DNA were separated by native-Tris-borate EDTA (TBE) polyacrylamide gel electrophoresis, stained with (B) toluidine blue, and (C) G4-specific dye N -methyl mesoporphyrin IX alongside their positive (G4: hTR 1-17 ) and negative (double stranded RNA) controls.
Figure Legend Snippet: Q2RNA, but not Q2DNA, stains with a parallel G4 dye. (A) Schematic showing the G4 forming regions of PITX1 mRNA; the RNA and DNA equivalent sequences with the guanylate tracts underlined are also shown. 250 pmol of Q2RNA and Q2DNA were separated by native-Tris-borate EDTA (TBE) polyacrylamide gel electrophoresis, stained with (B) toluidine blue, and (C) G4-specific dye N -methyl mesoporphyrin IX alongside their positive (G4: hTR 1-17 ) and negative (double stranded RNA) controls.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining

RHAU 53-105 forms a complex with Q2RNA. (A) Electrophoretic mobility shift assays (EMSA) were performed using a constant 150 nM concentration of Q2RNA or Q2DNA and a variable concentration from 0–700 nM of RHAU 53-105 or full-length RHAU. The 12% native Tris borate-EDTA (TBE) polyacrylamide gels were stained with SYBR Gold for visualization. (B) Microscale thermophoresis measurements performed using 3’-FAM Q2RNA (25 nM) in complex with RHAU 53-105 at several concentrations (0.6–250 nM). Reverse T-Jump signals from the traces were fit as described in the Materials Methods.
Figure Legend Snippet: RHAU 53-105 forms a complex with Q2RNA. (A) Electrophoretic mobility shift assays (EMSA) were performed using a constant 150 nM concentration of Q2RNA or Q2DNA and a variable concentration from 0–700 nM of RHAU 53-105 or full-length RHAU. The 12% native Tris borate-EDTA (TBE) polyacrylamide gels were stained with SYBR Gold for visualization. (B) Microscale thermophoresis measurements performed using 3’-FAM Q2RNA (25 nM) in complex with RHAU 53-105 at several concentrations (0.6–250 nM). Reverse T-Jump signals from the traces were fit as described in the Materials Methods.

Techniques Used: Electrophoretic Mobility Shift Assay, Concentration Assay, Staining, Microscale Thermophoresis

Normalized thermal difference spectra of Q2RNA and DNA counterpart. TDS of Q2RNA (circles) and Q2DNA (squares) in 10 mM Tris, pH 7.5, 100 mM KCl, 1 mM EDTA. For details of analysis, see Materials and Methods .
Figure Legend Snippet: Normalized thermal difference spectra of Q2RNA and DNA counterpart. TDS of Q2RNA (circles) and Q2DNA (squares) in 10 mM Tris, pH 7.5, 100 mM KCl, 1 mM EDTA. For details of analysis, see Materials and Methods .

Techniques Used:

32) Product Images from "Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells *"

Article Title: Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.663427

Basal expression from P frmRA -frmRE64H is higher than P frmRA -frmR . β-Galactosidase activity in Δ frmR containing P frmRA -frmR ( filled circles ) or P frmRA -frmRE64H ( open circles ) following growth to early exponential phase in the presence of EDTA ( A ) or TPEN ( B ). C , expression from P zntA in wild type Salmonella , grown as described in A , or D , as described in B. E , expression in Δ frmR containing P frmRA -frmR ( white bars ), P frmRA -frmR DOWN ( dashed white bars ), P frmRA -frmRE64H ( gray bars ), or P frmRA -frmRE64H UP ( dashed gray bars ) following growth to early exponential phase, and F , following exposure (2 h) to Zn(II), Co(II) or formaldehyde, or untreated control. G–J , multiple reaction monitoring, quantitative MS of cell extracts. Representative ( n = 3) extracted LC-MS chromatograms of ion transitions detected in Δ frmR containing P frmRA -frmR ( G ), P frmRA -frmR DOWN ( H ), P frmRA -frmRE64H ( I ), or P frmRA -frmRE64H UP ( J ). Transitions 451.24/716.4 and 456.24/726.4 are for analyte GQVEALER ( solid lines ) or labeled GQVEALE R [ 13 C 6 , 15 N 4 ] ([ 13 C 6 , 15 N 4 ]arginine residue) ( dashed lines ). K , abundance of FrmR and variants using quantitative data obtained in G–J .
Figure Legend Snippet: Basal expression from P frmRA -frmRE64H is higher than P frmRA -frmR . β-Galactosidase activity in Δ frmR containing P frmRA -frmR ( filled circles ) or P frmRA -frmRE64H ( open circles ) following growth to early exponential phase in the presence of EDTA ( A ) or TPEN ( B ). C , expression from P zntA in wild type Salmonella , grown as described in A , or D , as described in B. E , expression in Δ frmR containing P frmRA -frmR ( white bars ), P frmRA -frmR DOWN ( dashed white bars ), P frmRA -frmRE64H ( gray bars ), or P frmRA -frmRE64H UP ( dashed gray bars ) following growth to early exponential phase, and F , following exposure (2 h) to Zn(II), Co(II) or formaldehyde, or untreated control. G–J , multiple reaction monitoring, quantitative MS of cell extracts. Representative ( n = 3) extracted LC-MS chromatograms of ion transitions detected in Δ frmR containing P frmRA -frmR ( G ), P frmRA -frmR DOWN ( H ), P frmRA -frmRE64H ( I ), or P frmRA -frmRE64H UP ( J ). Transitions 451.24/716.4 and 456.24/726.4 are for analyte GQVEALER ( solid lines ) or labeled GQVEALE R [ 13 C 6 , 15 N 4 ] ([ 13 C 6 , 15 N 4 ]arginine residue) ( dashed lines ). K , abundance of FrmR and variants using quantitative data obtained in G–J .

Techniques Used: Expressing, Activity Assay, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling

Zn(II) weakens K DNA of FrmR and FrmRE64H and its effect on DNA occupancy. Anisotropy change upon titration of a high concentration of frmRA Pro (2.5 μ m ) with FrmR ( A ), FrmRE64H ( B ), or a limiting concentration of frmRA Pro (10 n m ) ( C ) with apo-FrmR in the presence of 5 m m EDTA ( closed symbols ) or Zn(II)-FrmR in the presence of 5 μ m ZnCl 2 ( open symbols ). D , as C but using FrmRE64H. Symbol shapes represent individual experiments. Data were fit to a model describing a 2:1 protein tetramer (nondissociable):DNA stoichiometry (binding with equal affinity), and lines represent simulated curves produced from the average K DNA determined across the experimental replicas shown. E , coupled thermodynamic equilibria (assuming a closed system) describing the relationship between FrmR tetramer ( P ), Zn(II) ( Z ), and P frmRA ( D ) ( 9 , 65 , 66 ). The coupling constant ( K C ) is determined from the ratio K 4 / K 3 ( K DNA Zn(II)·FrmR / K DNA FrmR ) ( Equation 1 ) and used to calculate K 2 (the Zn(II) affinity of the DNA-bound protein, K Zn(II) FrmR·DNA ) from K 1 (K Zn(II) FrmR ) (Equation 2). F, calculated fractional occupancy of P frmRA with FrmR ( filled circles ) and FrmRE64H ( open circles ) as a function of (buffered) [Zn(II)], which incorporates the determined FrmR or FrmRE64H abundance, K Zn(II) sensor (off DNA), and K DNA ( Table 1 ). Additional lines represent hypothetical fractional occupancy of P frmRA with FrmRE64H but substituting K Zn(II) ( dotted ) or K DNA ( dashed ) for that of FrmR. G, as F but using the determined abundance for FrmR DOWN ( solid symbols ) and FrmRE64H UP ( open symbols ). H and I , as F and G , respectively, except using K Zn(II) sensor·DNA (on-DNA) (calculated using the equations in E ).
Figure Legend Snippet: Zn(II) weakens K DNA of FrmR and FrmRE64H and its effect on DNA occupancy. Anisotropy change upon titration of a high concentration of frmRA Pro (2.5 μ m ) with FrmR ( A ), FrmRE64H ( B ), or a limiting concentration of frmRA Pro (10 n m ) ( C ) with apo-FrmR in the presence of 5 m m EDTA ( closed symbols ) or Zn(II)-FrmR in the presence of 5 μ m ZnCl 2 ( open symbols ). D , as C but using FrmRE64H. Symbol shapes represent individual experiments. Data were fit to a model describing a 2:1 protein tetramer (nondissociable):DNA stoichiometry (binding with equal affinity), and lines represent simulated curves produced from the average K DNA determined across the experimental replicas shown. E , coupled thermodynamic equilibria (assuming a closed system) describing the relationship between FrmR tetramer ( P ), Zn(II) ( Z ), and P frmRA ( D ) ( 9 , 65 , 66 ). The coupling constant ( K C ) is determined from the ratio K 4 / K 3 ( K DNA Zn(II)·FrmR / K DNA FrmR ) ( Equation 1 ) and used to calculate K 2 (the Zn(II) affinity of the DNA-bound protein, K Zn(II) FrmR·DNA ) from K 1 (K Zn(II) FrmR ) (Equation 2). F, calculated fractional occupancy of P frmRA with FrmR ( filled circles ) and FrmRE64H ( open circles ) as a function of (buffered) [Zn(II)], which incorporates the determined FrmR or FrmRE64H abundance, K Zn(II) sensor (off DNA), and K DNA ( Table 1 ). Additional lines represent hypothetical fractional occupancy of P frmRA with FrmRE64H but substituting K Zn(II) ( dotted ) or K DNA ( dashed ) for that of FrmR. G, as F but using the determined abundance for FrmR DOWN ( solid symbols ) and FrmRE64H UP ( open symbols ). H and I , as F and G , respectively, except using K Zn(II) sensor·DNA (on-DNA) (calculated using the equations in E ).

Techniques Used: Titration, Concentration Assay, Binding Assay, Produced

33) Product Images from "Homeodomain-like DNA binding proteins control the haploid-to-diploid transition in Dictyostelium"

Article Title: Homeodomain-like DNA binding proteins control the haploid-to-diploid transition in Dictyostelium

Journal: Science Advances

doi: 10.1126/sciadv.1602937

Assessing the DNA binding activity of MatA and MatB. ( A ) EMSA experiments show that adding an increasing concentration of MatA to a dsDNA oligonucleotide causes the free DNA band to be progressively replaced by a broad smear, indicating (nonspecific) binding (see text). ( B ) Mutating residues Lys 72 and Lys 76 to Ala abolishes this interaction, implicating these residues in the mechanism of DNA binding. ( C ) Addition of MatB causes a similar pattern to that seen for the addition of MatA. ( D and E ) Electrostatic potential surface of MatA, omitting the tail regions for clarity. The orientation shown in (D) (same as in Fig. 1A ) shows the pronounced basic patch resulting from the conserved basic residues on the surface of helix 3. ( F ) Adding various lengths of dsDNA to samples of 15 N-labeled MatA causes peaks to shift in the 15 N- 1 H HSQC (heteronuclear single-quantum coherence) NMR spectrum; these CSPs can be plotted as a histogram ( G ) and mapped as a color ramp onto the lowest energy structure of MatA ( H ), shown in the same orientation as (D). This shows that many of the shifts map to the third helix, again implicating this region in direct interactions with the DNA; Leu 32 , which is N-terminal to the core folded domain, is also strongly affected. The NMR experiments used MatA (20 μM) and DNA (80 μM) in 25 mM phosphate (pH 6), 50 mM NaCl, and 50 μM EDTA. ppm, parts per million.
Figure Legend Snippet: Assessing the DNA binding activity of MatA and MatB. ( A ) EMSA experiments show that adding an increasing concentration of MatA to a dsDNA oligonucleotide causes the free DNA band to be progressively replaced by a broad smear, indicating (nonspecific) binding (see text). ( B ) Mutating residues Lys 72 and Lys 76 to Ala abolishes this interaction, implicating these residues in the mechanism of DNA binding. ( C ) Addition of MatB causes a similar pattern to that seen for the addition of MatA. ( D and E ) Electrostatic potential surface of MatA, omitting the tail regions for clarity. The orientation shown in (D) (same as in Fig. 1A ) shows the pronounced basic patch resulting from the conserved basic residues on the surface of helix 3. ( F ) Adding various lengths of dsDNA to samples of 15 N-labeled MatA causes peaks to shift in the 15 N- 1 H HSQC (heteronuclear single-quantum coherence) NMR spectrum; these CSPs can be plotted as a histogram ( G ) and mapped as a color ramp onto the lowest energy structure of MatA ( H ), shown in the same orientation as (D). This shows that many of the shifts map to the third helix, again implicating this region in direct interactions with the DNA; Leu 32 , which is N-terminal to the core folded domain, is also strongly affected. The NMR experiments used MatA (20 μM) and DNA (80 μM) in 25 mM phosphate (pH 6), 50 mM NaCl, and 50 μM EDTA. ppm, parts per million.

Techniques Used: Binding Assay, Activity Assay, Concentration Assay, Labeling, Nuclear Magnetic Resonance

34) Product Images from "Multiple Regulatory Domains of IRF-5 Control Activation, Cellular Localization, and Induction of Chemokines That Mediate Recruitment of T Lymphocytes"

Article Title: Multiple Regulatory Domains of IRF-5 Control Activation, Cellular Localization, and Induction of Chemokines That Mediate Recruitment of T Lymphocytes

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.16.5721-5740.2002

). Template input, amplification of the endogenous IFNA promoter region from DNA-protein complexes before immunoprecipitation. Immunoprecipitated (i.p.) DNA was resuspended in 60 μl of Tris-EDTA. Serial dilutions (1, 5, or 25 μl) were used as templates for PCR amplification to show that the response was in the linear range. Levels of IRF-5 (anti-Flag Ab) and IRF-3 protein in cell lysates as detected by Western blotting are shown. (B) Cooperation between IRF-3 and IRF-5 binding to IFNA1 VRE enhances IRF-5-induced IFNA1 expression. 2fTGH cells were cotransfected with IFNA1 SAP (1 μg) and IRF-3 (2 μg) or IRF-5 (2 μg) or IRF-3 (2 μg) and IRF-5 (2 μg). All transfections were performed with equal amounts of total DNA (5 μg); pUC19 was used as filler DNA. At 16 h posttransfection, cells were left uninfected or were infected with NDV for an additional 16 h, and SAP activity was measured as described in Materials and Methods. SAP activity is expressed after normalizing for β-galactosidase expression.
Figure Legend Snippet: ). Template input, amplification of the endogenous IFNA promoter region from DNA-protein complexes before immunoprecipitation. Immunoprecipitated (i.p.) DNA was resuspended in 60 μl of Tris-EDTA. Serial dilutions (1, 5, or 25 μl) were used as templates for PCR amplification to show that the response was in the linear range. Levels of IRF-5 (anti-Flag Ab) and IRF-3 protein in cell lysates as detected by Western blotting are shown. (B) Cooperation between IRF-3 and IRF-5 binding to IFNA1 VRE enhances IRF-5-induced IFNA1 expression. 2fTGH cells were cotransfected with IFNA1 SAP (1 μg) and IRF-3 (2 μg) or IRF-5 (2 μg) or IRF-3 (2 μg) and IRF-5 (2 μg). All transfections were performed with equal amounts of total DNA (5 μg); pUC19 was used as filler DNA. At 16 h posttransfection, cells were left uninfected or were infected with NDV for an additional 16 h, and SAP activity was measured as described in Materials and Methods. SAP activity is expressed after normalizing for β-galactosidase expression.

Techniques Used: Amplification, Immunoprecipitation, Polymerase Chain Reaction, Western Blot, Binding Assay, Expressing, Transfection, Infection, Activity Assay

35) Product Images from "The electron distribution in the “activated” state of cytochrome c oxidase"

Article Title: The electron distribution in the “activated” state of cytochrome c oxidase

Journal: Scientific Reports

doi: 10.1038/s41598-018-25779-w

Absorbance difference spectra. A comparison of reduced minus oxidized difference spectra of the Cyt c O-cyt. c complex (black) and the sum of the reduced minus oxidized difference spectra of Cyt c O and cyt. c (red), respectively. Spectra of the oxidized states were recorded first and then the atmosphere in the cuvette was replaced for N 2 after which the samples were reduced with ascorbate (2 mM) and hexaammineruthenium(II) chloride (1 μM). Reduction of the samples was followed in time by recording spectra over ~2 hours until no further changes were observed. The data to the right of the axis break have been multiplied by a factor of five. The concentrations of Cyt c O and cyt. c were ~3 μM and ~5 μM, respectively, in 50 mM HEPES (pH 7.5), 0.05% DDM and 100 µM EDTA.
Figure Legend Snippet: Absorbance difference spectra. A comparison of reduced minus oxidized difference spectra of the Cyt c O-cyt. c complex (black) and the sum of the reduced minus oxidized difference spectra of Cyt c O and cyt. c (red), respectively. Spectra of the oxidized states were recorded first and then the atmosphere in the cuvette was replaced for N 2 after which the samples were reduced with ascorbate (2 mM) and hexaammineruthenium(II) chloride (1 μM). Reduction of the samples was followed in time by recording spectra over ~2 hours until no further changes were observed. The data to the right of the axis break have been multiplied by a factor of five. The concentrations of Cyt c O and cyt. c were ~3 μM and ~5 μM, respectively, in 50 mM HEPES (pH 7.5), 0.05% DDM and 100 µM EDTA.

Techniques Used:

Kinetics of absorbance changes upon reaction with O 2 . The four-electron reduced Cyt c O (black traces) or the five-electron-reduced cyt. c -Cyt c O complex (red traces) was mixed with an O 2 -saturated buffer solution. About 200 ms after mixing with the O 2 -containing buffer, at time = 0, the CO-ligand was removed by a laser flash. The four-electron complex becomes oxidized over a time scale of 8 ms (left-hand side boxes). At 445 nm ( a ) the main contribution is from redox changes at hemes a and a 3 . At 605 nm ( b ) the main contribution is from redox changes at heme a (80%) and the remaining fraction originates from changes at heme a 3 . At 830 nm ( c ) the main contribution is from Cu A where an increase in absorbance is associated with oxidation. At 550 nm ( d ) the main contribution is from redox changes at cyt. c . At 445 nm and 605 nm, the rapid change in absorbance at t = 0 is associated with CO dissociation. It is followed in time by a decrease in absorbance associated with binding of O 2 (τ ≅ 10 μs), formation of the P R state (τ ≅ 40 μs) and oxidation of the Cyt c O (τ ≅ 1.5 ms). The P R → F reaction is not seen at these wavelengths. At 830 nm two components are seen with time constants of 200 µs ( P R → F ) and 1.5 ms ( F → O ). Oxidation of cyt. c occurs over the same time scale. In the presence of cyt. c absorbance changes attributed to the Cyt c O (panels a–c) were smaller because the redox sites were re-reduced by cyt. c during the course of the reaction. Over a time scale of ~500 ms (right-hand side boxes) all redox sites become oxidized. The small increase in absorbance over this time scale in the absence of cyt. c is due to small fractional re-reduction of Cyt c O by ascorbate. Experimental conditions after mixing: 1.1 μM Cyt c O, 20 mM HEPES at pH 7.5, 0.05% DDM, 100 μM EDTA, 2 mM ascorbate, 1 μM hexa-ammine-ruthenium(II) chloride, 1 mM O 2 at ~22 °C (black trace). The red traces are averages of three traces obtained under the following conditions: [cyt. c ]/[Cyt c O] in μM ( i ) 2.1/1.6, ( ii ) 1.5/1.3, ( iii ) 1.7/1.3. The mixing ratio was 1:5 with an oxygen-saturated buffer solution (20 mM HEPES at pH 7.5). All traces have been scaled to 1 μM reacting Cyt c O based on the rapid change in absorbance at 445 nm at t = 0. A laser artifact at t = 0 has been truncated for clarity.
Figure Legend Snippet: Kinetics of absorbance changes upon reaction with O 2 . The four-electron reduced Cyt c O (black traces) or the five-electron-reduced cyt. c -Cyt c O complex (red traces) was mixed with an O 2 -saturated buffer solution. About 200 ms after mixing with the O 2 -containing buffer, at time = 0, the CO-ligand was removed by a laser flash. The four-electron complex becomes oxidized over a time scale of 8 ms (left-hand side boxes). At 445 nm ( a ) the main contribution is from redox changes at hemes a and a 3 . At 605 nm ( b ) the main contribution is from redox changes at heme a (80%) and the remaining fraction originates from changes at heme a 3 . At 830 nm ( c ) the main contribution is from Cu A where an increase in absorbance is associated with oxidation. At 550 nm ( d ) the main contribution is from redox changes at cyt. c . At 445 nm and 605 nm, the rapid change in absorbance at t = 0 is associated with CO dissociation. It is followed in time by a decrease in absorbance associated with binding of O 2 (τ ≅ 10 μs), formation of the P R state (τ ≅ 40 μs) and oxidation of the Cyt c O (τ ≅ 1.5 ms). The P R → F reaction is not seen at these wavelengths. At 830 nm two components are seen with time constants of 200 µs ( P R → F ) and 1.5 ms ( F → O ). Oxidation of cyt. c occurs over the same time scale. In the presence of cyt. c absorbance changes attributed to the Cyt c O (panels a–c) were smaller because the redox sites were re-reduced by cyt. c during the course of the reaction. Over a time scale of ~500 ms (right-hand side boxes) all redox sites become oxidized. The small increase in absorbance over this time scale in the absence of cyt. c is due to small fractional re-reduction of Cyt c O by ascorbate. Experimental conditions after mixing: 1.1 μM Cyt c O, 20 mM HEPES at pH 7.5, 0.05% DDM, 100 μM EDTA, 2 mM ascorbate, 1 μM hexa-ammine-ruthenium(II) chloride, 1 mM O 2 at ~22 °C (black trace). The red traces are averages of three traces obtained under the following conditions: [cyt. c ]/[Cyt c O] in μM ( i ) 2.1/1.6, ( ii ) 1.5/1.3, ( iii ) 1.7/1.3. The mixing ratio was 1:5 with an oxygen-saturated buffer solution (20 mM HEPES at pH 7.5). All traces have been scaled to 1 μM reacting Cyt c O based on the rapid change in absorbance at 445 nm at t = 0. A laser artifact at t = 0 has been truncated for clarity.

Techniques Used: Mass Spectrometry, Binding Assay

Reaction of membrane fragments with O 2 . The experiment is the same as that shown in Fig. 3 . Absorbance changes were monitored at 445 nm ( a ) 605 nm ( b ) and 550 nm ( c ) Experimental conditions after mixing: R. sphaeroides membranes (∼2 µM Cyt c O, calculated from the reduced minus oxidized difference spectrum) in 20 mM HEPES (pH 7.5), 100 µM EDTA and 1 mM O 2 at ∼22 °C. The mixing ratio was 1:1 with an oxygen-saturated buffer (20 mM HEPES pH 7.5). The traces have been scaled to 1 µM reacting Cyt c O. A laser artifact at t = 0 has been truncated for clarity.
Figure Legend Snippet: Reaction of membrane fragments with O 2 . The experiment is the same as that shown in Fig. 3 . Absorbance changes were monitored at 445 nm ( a ) 605 nm ( b ) and 550 nm ( c ) Experimental conditions after mixing: R. sphaeroides membranes (∼2 µM Cyt c O, calculated from the reduced minus oxidized difference spectrum) in 20 mM HEPES (pH 7.5), 100 µM EDTA and 1 mM O 2 at ∼22 °C. The mixing ratio was 1:1 with an oxygen-saturated buffer (20 mM HEPES pH 7.5). The traces have been scaled to 1 µM reacting Cyt c O. A laser artifact at t = 0 has been truncated for clarity.

Techniques Used:

36) Product Images from "Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W"

Article Title: Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

Journal:

doi: 10.1016/j.abb.2006.12.025

Analytical ultracentrifugation data. (a) The sedimentation profiles for 1.0 μM CYP3A4 WT, 1.0 μM FEW (1), and 1.0 μM FEW (2). The buffer contained 0.05 M Na-HEPES buffer (pH 7.4), 0.5 mM DTT, 0.5 mM EDTA and 10% glycerol. The temperature
Figure Legend Snippet: Analytical ultracentrifugation data. (a) The sedimentation profiles for 1.0 μM CYP3A4 WT, 1.0 μM FEW (1), and 1.0 μM FEW (2). The buffer contained 0.05 M Na-HEPES buffer (pH 7.4), 0.5 mM DTT, 0.5 mM EDTA and 10% glycerol. The temperature

Techniques Used: Sedimentation

Kinetics of dithionite-dependent reduction of CYP3A4 WT and FEW in solution. Conditions: 3 μM 3A4, 12.5 mM sodium dithionite, CO-saturated 0.1 M Na-HEPES buffer (pH = 7.4), 1 mM DTT, 1 mM EDTA, 25 °C. Spectra recorded in a stop-flow cell
Figure Legend Snippet: Kinetics of dithionite-dependent reduction of CYP3A4 WT and FEW in solution. Conditions: 3 μM 3A4, 12.5 mM sodium dithionite, CO-saturated 0.1 M Na-HEPES buffer (pH = 7.4), 1 mM DTT, 1 mM EDTA, 25 °C. Spectra recorded in a stop-flow cell

Techniques Used: Flow Cytometry

37) Product Images from "Oxygen-Linked S-Nitrosation in Fish Myoglobins: A Cysteine-Specific Tertiary Allosteric Effect"

Article Title: Oxygen-Linked S-Nitrosation in Fish Myoglobins: A Cysteine-Specific Tertiary Allosteric Effect

Journal: PLoS ONE

doi: 10.1371/journal.pone.0097012

S-nitrosation increases O 2 affinity of salmon and trout Mbs but not of tuna Mb and is functionally equivalent to modification by N -ethylmaleimide. A) O 2 equilibrium curves for tuna and salmon Mb and Mb-SNO and B) O 2 equilibrium curves for trout Mb, Mb-NEM and Mb-SNO, as indicated, measured in 50 mM Tris, 0.5 mM EDTA, pH 8.3 at 20°C. Mb-SNO data are from [9] .
Figure Legend Snippet: S-nitrosation increases O 2 affinity of salmon and trout Mbs but not of tuna Mb and is functionally equivalent to modification by N -ethylmaleimide. A) O 2 equilibrium curves for tuna and salmon Mb and Mb-SNO and B) O 2 equilibrium curves for trout Mb, Mb-NEM and Mb-SNO, as indicated, measured in 50 mM Tris, 0.5 mM EDTA, pH 8.3 at 20°C. Mb-SNO data are from [9] .

Techniques Used: Modification

38) Product Images from "The Arabidopsis Chloroplastic NifU-Like Protein CnfU, Which Can Act as an Iron-Sulfur Cluster Scaffold Protein, Is Required for Biogenesis of Ferredoxin and Photosystem I W⃞"

Article Title: The Arabidopsis Chloroplastic NifU-Like Protein CnfU, Which Can Act as an Iron-Sulfur Cluster Scaffold Protein, Is Required for Biogenesis of Ferredoxin and Photosystem I W⃞

Journal: The Plant Cell

doi: 10.1105/tpc.020511

Gel Filtration Analysis Revealed a Dimeric Holo-State and a Predominantly Monomeric Apo-State of AtCnfU-V. (A) Gel filtration chromatograms of holo- and apo-AtCnfU-V. After incubation with (dotted lines) or without (solid lines) 10 mM EDTA or 1 mM dithionite (dashed lines) on ice for 1 h, purified AtCnfU-V (25 μg) was applied to a Superdex 75 column (Amersham Biosciences) and equilibrated with buffer containing 50 mM Hepes-KOH, pH 7.5, 150 mM KCl, and 5 mM DTT. Eluates were monitored simultaneously by absorbance at 280 nm (top), 330 nm (middle), and 420 nm (bottom) and divided into 12 fractions. Molecular mass marker proteins used were BSA (67 kD), ovalbumin (43 kD), myoglobin (18 kD), and aprotinin (6.5 kD). ABS, absorbance. (B) Fractions (3 to 12) obtained from each gel filtration chromatography analysis shown in (A) were analyzed by protein gel blotting using an anti-AtCnfU-V antibody.
Figure Legend Snippet: Gel Filtration Analysis Revealed a Dimeric Holo-State and a Predominantly Monomeric Apo-State of AtCnfU-V. (A) Gel filtration chromatograms of holo- and apo-AtCnfU-V. After incubation with (dotted lines) or without (solid lines) 10 mM EDTA or 1 mM dithionite (dashed lines) on ice for 1 h, purified AtCnfU-V (25 μg) was applied to a Superdex 75 column (Amersham Biosciences) and equilibrated with buffer containing 50 mM Hepes-KOH, pH 7.5, 150 mM KCl, and 5 mM DTT. Eluates were monitored simultaneously by absorbance at 280 nm (top), 330 nm (middle), and 420 nm (bottom) and divided into 12 fractions. Molecular mass marker proteins used were BSA (67 kD), ovalbumin (43 kD), myoglobin (18 kD), and aprotinin (6.5 kD). ABS, absorbance. (B) Fractions (3 to 12) obtained from each gel filtration chromatography analysis shown in (A) were analyzed by protein gel blotting using an anti-AtCnfU-V antibody.

Techniques Used: Filtration, Incubation, Purification, Marker, Chromatography

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Chromatography:

Article Title: Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability
Article Snippet: .. After elution with 2 mM EDTA, gel exclusion chromatography with Superose 6 (Amersham Biosciences, Piscataway, New Jersey, USA) was used to remove contaminating SP-D. ..

Purification:

Article Title: Identification of Channel-lining Amino Acid Residues in the Hydrophobic Segment of Colicin Ia
Article Snippet: .. Dialyzed streptomycin-sulfate supernatants in 50 mM sodium borate, pH 9.0, 2 mM dithiothreitol (DTT), and 2 mM EDTA were purified on 1- or 5-ml pre-packed HiTrap CM FF columns (GE Healthcare). ..

Article Title: Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities
Article Snippet: .. For further purification, the buffer of partially purified enzymes was changed to 50 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA (pH 8.0) by using PD-10 columns (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). .. Final purification was obtained by the use of size exclusion chromatography on Superdex 200 column (GE Healthcare) equilibrated in 50 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA (pH 8.0).

Article Title: The Arabidopsis Chloroplastic NifU-Like Protein CnfU, Which Can Act as an Iron-Sulfur Cluster Scaffold Protein, Is Required for Biogenesis of Ferredoxin and Photosystem I W⃞
Article Snippet: .. Purified AtCnfU-V (25 μg) dissolved in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM KCl, and 5 mM DTT were further treated either with 10 mM EDTA or 1 mM DTT on ice for 1 h. After treatment, samples were loaded onto Superdex 75 columns (Amersham Biosciences) and eluted with the same buffer. ..

Fast Protein Liquid Chromatography:

Article Title: Stability and Function of the Sec61 Translocation Complex Depends on the Sss1p Tail-Anchor Sequence
Article Snippet: .. A total of 300µl of the cleared sample was loaded directly onto a Superdex 200 HR 10/30 column (or Superose 6 HR 10/30 column for WT, CTSa and TMSa samples) (Amersham Pharmacia Biotech, GE Healthcare Life Sciences, Canada) pre-equilibrated in 50mM HEPES-KOH, pH 7.5, 500mM potassium acetate, 0.1% digitonin, 1mM EDTA, 5mM β-mercaptoethanol, and 1XCPIC and run at 0.2ml/min on an ÄKTA FPLC System (Amersham Pharmacia Biotech, GE Healthcare Life Sciences, Canada) at 4°C. .. Fifty 0.5ml fractions were collected, and the proteins were precipitated with trichloroacetic acid, separated by SDS-PAGE, and transferred to nitrocellulose.

Article Title: Oxygen-Linked S-Nitrosation in Fish Myoglobins: A Cysteine-Specific Tertiary Allosteric Effect
Article Snippet: .. Samples were centrifuged for 25 min at 12,000 g, passed on a PD-10 column (GE Healthcare) equilibrated with 50 mM Tris-HCl, 0.5 mM EDTA, 3 mM dithiothreitol (DTT), pH 8.3 and loaded on a gel-filtration Tricorn Superdex 75 10/300 GL FPLC-column (GE-Healthcare) equilibrated with 50 mM Tris-HCl, 0.5 mM EDTA, 3 mM DTT, 0.15 M NaCl, pH 8.3. .. Finally, the sample was dialysed against 20 mM Tris-HCl pH 9.2, passed through an ion exchange Hitrap Q-FF FPLC-column, and eluted with a 30-min gradient of 0–0.5 M NaCl in 20 mM Tris-HCl pH 9.2 at a flow rate of 1 mL/min.

Western Blot:

Article Title: A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1
Article Snippet: .. Western blotting analysis The Hpn protein (25 μM) treated with indicated mol equivalent of EDTA or Ni2+ resolved on SDS-PAGE (20%) and then electrophoretically blotted onto polyvinylidene fluoride (PVDF) membrane (Amersham Hybond-P, GE Healthcare, code: RPN303F) in transfer buffer containing no methanol. ..

Recombinant:

Article Title: DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells
Article Snippet: .. The DSS1-V5-His recombinant protein was eluted with 1× TBS [20 mmol/L Tris-HCl (pH = 7.4) and 0.9% NaCl] containing 50 mmol/L EDTA followed by loading it onto the 1 mL HiTrap Capto DEAE ion exchange column (GE Healthcare). ..

SDS Page:

Article Title: A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1
Article Snippet: .. Western blotting analysis The Hpn protein (25 μM) treated with indicated mol equivalent of EDTA or Ni2+ resolved on SDS-PAGE (20%) and then electrophoretically blotted onto polyvinylidene fluoride (PVDF) membrane (Amersham Hybond-P, GE Healthcare, code: RPN303F) in transfer buffer containing no methanol. ..

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    GE Healthcare edta plasma supernatant
    Separation of C3dg from the larger C3 fragments. Measurements of C3 molecules encompassing the determinants of the segment C3dg, i.e., C3 and all degradation molecules containing this part of C3. (A) The figure illustrates C3dg measurement on gel permeation chromatography (GPC) fractions of serum before activation (red line) and supernatant of polyethylene glycol <t>(PEG)-precipitated</t> activated serum (blue line). (B) Test for the optimal concentration of PEG for precipitation. Increasing concentrations of PEG were added to <t>EDTA</t> plasma and C3dg was estimated in the supernatants. (C) GPC of supernatant after precipitation of EDTA plasma with 11% (red) and 16% (blue) PEG. C3dg in the fractions was measured. The 11% supernatant shows two major peaks, the first corresponding to C3 and larger C3 components, and the second corresponding to free C3dg. After precipitation with 16% PEG the first was significantly reduced. Panel (D) shows the results of western blotting of supernatants of EDTA-plasma precipitated with 16% (lane 1) or 11% (lane 2) PEG. The samples (corresponding to 0.1 µl plasma) were run non-reduced on the SDS-PAGE. The blot was developed with anti-C3d antibody. It can be seen that the 11% PEG supernatant still contains appreciable amounts of larger C3dg-encompassing molecules. This was repeated three times with similar results.
    Edta Plasma Supernatant, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare edta
    Steady-state kinetic parameters for mAOX1-4 with aromatic aldehydes as substrates containing a benzyl-group. Apparent steady-state kinetic parameters were recorded in 50 mM <t>Tris-HCl,</t> 200 mM NaCl, and 1 mM <t>EDTA</t> (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
    Edta, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare cr 51 edta stock solution
    Improvement in gamma camera-based GFR measurements using the revised equation. The GFRs from Gates’ original equation of [GFR(mL/min) = ( % renal uptake × 9.8127) − 6.82519] were significantly lower than those from the <t>Cr-51</t> <t>EDTA</t> test ( P
    Cr 51 Edta Stock Solution, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Separation of C3dg from the larger C3 fragments. Measurements of C3 molecules encompassing the determinants of the segment C3dg, i.e., C3 and all degradation molecules containing this part of C3. (A) The figure illustrates C3dg measurement on gel permeation chromatography (GPC) fractions of serum before activation (red line) and supernatant of polyethylene glycol (PEG)-precipitated activated serum (blue line). (B) Test for the optimal concentration of PEG for precipitation. Increasing concentrations of PEG were added to EDTA plasma and C3dg was estimated in the supernatants. (C) GPC of supernatant after precipitation of EDTA plasma with 11% (red) and 16% (blue) PEG. C3dg in the fractions was measured. The 11% supernatant shows two major peaks, the first corresponding to C3 and larger C3 components, and the second corresponding to free C3dg. After precipitation with 16% PEG the first was significantly reduced. Panel (D) shows the results of western blotting of supernatants of EDTA-plasma precipitated with 16% (lane 1) or 11% (lane 2) PEG. The samples (corresponding to 0.1 µl plasma) were run non-reduced on the SDS-PAGE. The blot was developed with anti-C3d antibody. It can be seen that the 11% PEG supernatant still contains appreciable amounts of larger C3dg-encompassing molecules. This was repeated three times with similar results.

    Journal: Frontiers in Immunology

    Article Title: The C3dg Fragment of Complement Is Superior to Conventional C3 as a Diagnostic Biomarker in Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.00581

    Figure Lengend Snippet: Separation of C3dg from the larger C3 fragments. Measurements of C3 molecules encompassing the determinants of the segment C3dg, i.e., C3 and all degradation molecules containing this part of C3. (A) The figure illustrates C3dg measurement on gel permeation chromatography (GPC) fractions of serum before activation (red line) and supernatant of polyethylene glycol (PEG)-precipitated activated serum (blue line). (B) Test for the optimal concentration of PEG for precipitation. Increasing concentrations of PEG were added to EDTA plasma and C3dg was estimated in the supernatants. (C) GPC of supernatant after precipitation of EDTA plasma with 11% (red) and 16% (blue) PEG. C3dg in the fractions was measured. The 11% supernatant shows two major peaks, the first corresponding to C3 and larger C3 components, and the second corresponding to free C3dg. After precipitation with 16% PEG the first was significantly reduced. Panel (D) shows the results of western blotting of supernatants of EDTA-plasma precipitated with 16% (lane 1) or 11% (lane 2) PEG. The samples (corresponding to 0.1 µl plasma) were run non-reduced on the SDS-PAGE. The blot was developed with anti-C3d antibody. It can be seen that the 11% PEG supernatant still contains appreciable amounts of larger C3dg-encompassing molecules. This was repeated three times with similar results.

    Article Snippet: Samples of serum, supernatant of PEG precipitated activated serum and EDTA-plasma supernatant after precipitation with either 11 or 16% PEG (w/v), were subjected to GPC on a Superose 6 10/300 GL column (GE Healthcare).

    Techniques: GPC Assay, Gel Permeation Chromatography, Activation Assay, Concentration Assay, Western Blot, SDS Page

    Steady-state kinetic parameters for mAOX1-4 with aromatic aldehydes as substrates containing a benzyl-group. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

    Journal: PLoS ONE

    Article Title: Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities

    doi: 10.1371/journal.pone.0191819

    Figure Lengend Snippet: Steady-state kinetic parameters for mAOX1-4 with aromatic aldehydes as substrates containing a benzyl-group. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

    Article Snippet: For further purification, the buffer of partially purified enzymes was changed to 50 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA (pH 8.0) by using PD-10 columns (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).

    Techniques: Variant Assay, Activity Assay

    Steady-state kinetic parameters for mAOX1-4 with N-heterocyclic compounds as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

    Journal: PLoS ONE

    Article Title: Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities

    doi: 10.1371/journal.pone.0191819

    Figure Lengend Snippet: Steady-state kinetic parameters for mAOX1-4 with N-heterocyclic compounds as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

    Article Snippet: For further purification, the buffer of partially purified enzymes was changed to 50 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA (pH 8.0) by using PD-10 columns (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).

    Techniques: Variant Assay, Activity Assay

    Steady-state kinetic parameters for mAOX1-4 with aliphatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

    Journal: PLoS ONE

    Article Title: Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities

    doi: 10.1371/journal.pone.0191819

    Figure Lengend Snippet: Steady-state kinetic parameters for mAOX1-4 with aliphatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

    Article Snippet: For further purification, the buffer of partially purified enzymes was changed to 50 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA (pH 8.0) by using PD-10 columns (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).

    Techniques: Variant Assay, Activity Assay

    Steady-state kinetic parameters for mAOX1-4 with cinnamaldehyde-related compounds as substrates aromatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

    Journal: PLoS ONE

    Article Title: Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities

    doi: 10.1371/journal.pone.0191819

    Figure Lengend Snippet: Steady-state kinetic parameters for mAOX1-4 with cinnamaldehyde-related compounds as substrates aromatic aldehydes as substrates. Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the K M . The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.

    Article Snippet: For further purification, the buffer of partially purified enzymes was changed to 50 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA (pH 8.0) by using PD-10 columns (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).

    Techniques: Variant Assay, Activity Assay

    Effect of trans MTSET on the macroscopic current through colicin Ia mutant N578C channels. The top trace shows the membrane current and the bottom trace shows the voltage, each as a function of time. Before the start of the record, DTT was added to the trans compartment to a final concentration of 5 μM and 1.0 μg N578C (along with 4.5 μg octylglucoside and DTT to 5 μM) was added to the cis compartment. We quickly opened on the order of 1,000 channels by stepping the membrane potential to +70 mV, and then switched it to +50 mV to establish a slower channel-opening rate. At the arrow, 200 μg MTSET was added to the trans compartment. This caused a decrease in current of ∼25%, demonstrating that residue N578C was accessible for reaction. Finally, we confirmed that the channels closed normally when the membrane potential was switched to −50 mV. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM HEPES, pH 7.2. (The membrane broke after colicin addition and was reformed before the start of the record; similar results were obtained with membranes that had not broken.)

    Journal: The Journal of General Physiology

    Article Title: Identification of Channel-lining Amino Acid Residues in the Hydrophobic Segment of Colicin Ia

    doi: 10.1085/jgp.200810042

    Figure Lengend Snippet: Effect of trans MTSET on the macroscopic current through colicin Ia mutant N578C channels. The top trace shows the membrane current and the bottom trace shows the voltage, each as a function of time. Before the start of the record, DTT was added to the trans compartment to a final concentration of 5 μM and 1.0 μg N578C (along with 4.5 μg octylglucoside and DTT to 5 μM) was added to the cis compartment. We quickly opened on the order of 1,000 channels by stepping the membrane potential to +70 mV, and then switched it to +50 mV to establish a slower channel-opening rate. At the arrow, 200 μg MTSET was added to the trans compartment. This caused a decrease in current of ∼25%, demonstrating that residue N578C was accessible for reaction. Finally, we confirmed that the channels closed normally when the membrane potential was switched to −50 mV. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM HEPES, pH 7.2. (The membrane broke after colicin addition and was reformed before the start of the record; similar results were obtained with membranes that had not broken.)

    Article Snippet: Dialyzed streptomycin-sulfate supernatants in 50 mM sodium borate, pH 9.0, 2 mM dithiothreitol (DTT), and 2 mM EDTA were purified on 1- or 5-ml pre-packed HiTrap CM FF columns (GE Healthcare).

    Techniques: IA, Mutagenesis, Concentration Assay

    Improvement in gamma camera-based GFR measurements using the revised equation. The GFRs from Gates’ original equation of [GFR(mL/min) = ( % renal uptake × 9.8127) − 6.82519] were significantly lower than those from the Cr-51 EDTA test ( P

    Journal: European Radiology

    Article Title: Improved measurement of the glomerular filtration rate from Tc-99m DTPA scintigraphy in patients following nephrectomy

    doi: 10.1007/s00330-013-3039-z

    Figure Lengend Snippet: Improvement in gamma camera-based GFR measurements using the revised equation. The GFRs from Gates’ original equation of [GFR(mL/min) = ( % renal uptake × 9.8127) − 6.82519] were significantly lower than those from the Cr-51 EDTA test ( P

    Article Snippet: Cr-51 EDTA stock solution (37 MBq/10 mL, GE Healthcare) was diluted 1:10 with normal saline.

    Techniques: